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The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

Abstract

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Introduction

Most intracellular functions are carried out by proteins, assembled as chains of peptide-bond linked amino acid (aa) residues on large ribonucleoprotein particles called ribosomes. The aa-sequences are specified by information stored as deoxyribonucleic acid (DNA) sequences in the genome and transcribed into sequences of messenger RNAs (mRNAs). The mRNAs are translated into aa-sequences with the help of transfer RNAs (tRNAs) reading any of their 61 aa-encoding ribonucleotide triplets (codons). In termination of translation, the complete protein is released from the ribosome by a class-1 release factor (RF) recognizing one of the universal stop codons (UAA, UAG, and UGA), signaling the end of the amino acid encoding open reading frame (ORF) of the mRNA. There are two RFs in bacteria, RF1 and RF2, one in eukarya, eRF1. RF1 and RF2 read UAA, UAG, and UAA, UGA, respectively, while the omnipotent eRF1 reads all three stop codons. Each stop codon in the decoding center (DC) is recognized by a stop-codon recognition (SCR) motif in a class-1 RF, and all RFs have a peptidyl transfer center (PTC)-binding GGQ motif, named after its universal Gly–Gly–Gln triplet (GGQ), for coordinated ester bond hydrolysis in the P-site bound peptidyl-tRNA. The crystal structures of free RF1 and RF2 have a distance between the SCR and GGQ motifs of about 20 Å1,2, much shorter than the 70 Å separating DC and PTC in the bacterial 70S ribosome. This distance discrepancy made the expected coordination between SCR and ester bond hydrolysis enigmatic. The crystal structure of free eRF1 has, in contrast, about 70 Å between its SCR and GGQ motifs, a distance close to the 80 Å between the DC and PTC of the 80S ribosome in eukarya3. Further cryo-EM work showed that ribosome-bound RF1 and RF2 have extended structures4,5, facilitating coordinated codon recognition in DC and ester bond hydrolysis in PTC. Subsequent high-resolution X-ray crystal6,7,8,9,10,11,12,13 and cryo-EM14,15,16,17,18,19 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF220, eRF121, and GGQ-mediated ester bond hydrolysis22.

If the compact forms of free RFs in the crystal1,2 are physiologically relevant, it would mean that eubacterial RFs are in the compact form upon A-site entry (pre-accommodation state) and assume the extended form (accommodation state) in a stop-codon dependent manner. The relevance is indicated by a compact crystal structure of RF1 in a functional complex with its GGQ-modifying methyltransferase23,24, although SAXS data indicated free RF1 to be extended in bulk solution25. At the same time, SAXS data from T. thermophilus RF2 free in solution suggested a compact form for the factor or, possibly a mixture of compact and extended forms26. The existence of a RF-switch from a compact, free form to an extended ribosome-bound from would make high-resolution structures of these RF-forms necessary for a correct description of the stop-codon recognition process, hitherto based on post-termination ribosomal complexes27,28.

Indirect evidence for rapid conformational activation of RF1 and RF2 after A-site binding has been provided by quench-flow based kinetics22, and in a series of recent FRET experiments Joseph and collaborators showed free RF1 to be in a compact form29, compatible with the crystal forms of RF11 and RF22, but in an extended form when bound to the A site of the stop-codon programmed ribosome29. Ribosome-bound class-1 RFs in the compact form has been observed together with alternative ribosome-rescue factor A (ArfA) in ribosomal rescue complexes, which lack any codon in the A site14,15. Very recently, Svidritskiy and Korostelev6 used X-ray crystallography in conjunction with the peptidyl transfer-inhibiting antibiotic blasticidin S (BlaS) to capture a mutated, hyper-accurate variant of RF1 in the stop codon-programmed termination complex. They found RF1 in a compact form, which they used to discuss stop-codon recognition in conjunction with large conformational changes of the RFs. It seems, however, that this BlaS-halted ribosomal complex is in a post-recognition state (i.e., stop-codon recognition motif has the same conformation as in the post-accommodation state in DC) but before RF-accommodation in the A site, making its relevance for on-pathway stop-codon recognition unclear (However, see also below!).

Here, in contrast, we use time-resolved cryo-EM30,31,32,33,34 for real-time monitoring of how RF1 and RF2 ensembles change from compact to extended RF conformation in the first 100 ms after RF-binding to the pre-termination ribosome. These compact RF-structures, originating from short-lived ribosomal complexes previously out of reach for structural analysis, are seen at near-atomic resolution (3.5–4 Å). The time-dependent ensemble changes agree qualitatively with accompanying and previous22 quench-flow studies. We discuss the role of the compact structures of RF1 and RF2 for fast and accurate stop-codon recognition in translation termination.

Results

Kinetics study predicts compact RF1/RF2 exist at 20 ms

We assembled a UAA-programmed release complex, RC0, with tripeptidyl-tRNA in the P site, and visualized its structure with cryo-EM (Methods and Supplementary Fig. 1). The RC0 displays no intersubunit rotation, and the tripeptide of its P-site tRNA is seen near the end of the peptide exit tunnel (Supplementary Fig. 1). The mRNA of the DC is disordered, but the overall resolution of the RC0 is high (2.9 Å). Apart from a small fraction of isolated ribosomal 50S subunits, the RC0 ensemble is homogeneous (Supplementary Fig. 1). We used quench-flow techniques to monitor the time evolution of the class-1 RF-dependent release of tripeptide from the peptidyl-tRNA with UAA-codon in the A site after rapid mixing of RC0 with RF1 or RF2 at rate-saturating concentrations (kcat-range)22 (Fig. 1a). The experiments were performed at pH values from 6 to 8 units, corresponding to [OH] variation in the 0.25–2.5 µM range (Supplementary Figs 2 and  3). The results are consistent with the existence of a two-step mechanism, in which a pH-independent conformational change (rate constant kconf) is followed by pH-dependent ester bond hydrolysis (see Methods). We estimate kconf as 18 ± 3 s−1 for RF1 and 11 ± 1 s−1 for RF2 at 25 °C, which approximates the effective incubation temperature for the time-resolved cryo-EM experiments (Supplementary Figs 2 and  3).

Time evolution of ribosome ensembles in termination of translation a. Cartoon visualization of the pathway from free release complex to peptide release. Compact class 1 release factor (RF) binds to RF-free ribosomal release complex (RC0) and forms the RC·RFcompact complex with compounded rate constant ka·[RFfree]. Stop codon recognition induces conformational change in the RF which brings the ribosome from the RC·RFcompact to the RC·RFextended complex with rate constant kconf. The ester bond between the peptide and the P-site tRNA is hydrolyzed with rate constant khydr. b Predicted dynamics of peptide release with conformational change in RF1. We solved the ordinary differential equations associated with termination according to the scheme in a with association rate constant ka = 45 µM−1s−1, [RF1free] = 3 µM, kconf = 18 s−1 and khydr = 2 s−1 (Supplementary Fig. 2) and plotted the fractions of ribosomes in RC0, RC·RFcompact and RC·RFextended forms (y-axis) as functions of time (x-axis). Green dot lines, RC0; red solid lines, RC·RFcompact; blue dash lines, RC·RFextended. c Predicted dynamics of peptide release with conformational change in RF2. The fractions of ribosomes in different release complexes were obtained in the same way as b with the rate constants ka = 17 µM−1 s−1, [RF2free] = 3 µM, kconf = 11 s−1 and khydr = 2.7 s−1 (Supplementary Fig. 3). d, e The populations of release complexes containing compact conformation and extended conformation of RF1 (d) and RF2 (e) at the 24 ms, 60 ms and long incubation time points as obtained by time-resolved cryo-EM after 3D classification of the particle images

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From the quench-flow data, we predicted that the ensemble fraction of the compact RF1/2 form would be predominant at 24 ms, and much smaller at 60 ms (Fig. 1b, c). These predictions are in qualitative agreement with the time-resolved cryo-EM data, which show a somewhat faster conformational transition than in the quench-flow experiments (Fig. 1d, e). The difference in termination rates is, we suggest, due to a local temperature increase by friction inside the microfluidic chip. We first focus on the cryo-EM structures of RF1, and then highlight the few structural differences between RF1 and RF2.

Time-resolved cryo-EM analysis

At 24 ms reaction time, 25% of ribosome-bound RF1 is in the compact form in what we name the pre-accommodation state of the ribosome (Fig. 1d). The 70S part of the complex is similar to that of the pre-termination complex preceding RF-binding, but there is an additional A-site density belonging to RF1 (Fig. 2a). In pre-accommodation state of the ribosome, domain III of RF1 is 60–70 Å away from the PTC, in a similar relative orientation as in the crystal forms of the free factors1,2 (Supplementary Fig. 4) and significantly differing from that in the post-accommodated state of the terminating ribosome5. The loop that contains the GGQ motif of RF1 is positioned at the side of the β-sheet of domain II (near aa 165–168), facing the anticodon-stem loop and the D stem of the P-site tRNA (Fig. 2a, c).

Cryo-EM structures of E.coli 70S ribosome bound with release factor 1. a Pre-accommodated ribosome complex bound with RF1 in a compact conformation. b Accommodated ribosome complex bound with RF1 in an extended conformation. Light blue: 50S large subunit; light gold: 30S small subunit; green: tripeptide; orange: P-tRNA; pink: mRNA; red: compact RF1; and dark blue: extended RF1. c, d Positions of domain III of ribosome-bound RF1 in pre-accommodated ribosome complex (c) and accommodated RF1-ribosome complex (d) relative to mRNA (pink), P-tRNA (orange) and tripeptide (green). e, f Close-up views of the upper peptide exit tunnel, showing tripeptide (green) in pre-accommodated ribosome complex (e) and accommodated ribosome complex (f)

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At 60 ms reaction time the RF1-bound ribosome ensemble is dominated by the extended form of RF1 (Fig. 2b, d). We term the ribosome complex with extended RF1 the accommodated RF1-ribosome complex. It contains density for the tripeptide in the exit tunnel, indicating that at 60 ms the peptide has not been released from the ribosome (Fig. 2e, f and Supplementary Fig. 5). At a much later time-point (45 s) the tripeptide density is no longer present in the exit tunnel of the accommodated RF-ribosome complex. Precise estimation of the time evolution of tripeptide dissociation from the ribosome would require additional time points. Of particular functional relevance would be estimates of the time of dissociation of longer peptide chains from the exit tunnel.

The most striking difference between the compact and extended conformation of ribosome-bound RF1 is the position of the GGQ of domain III. As RF1 switches its conformation from the compact to the extended form, the repositioning of domain III places the catalytic GGQ motif within the PTC, and adjacent to the CCA end of the P-site tRNA (Fig. 2c, d). The extended form of RF1 has a similar conformation as found in the previous studies7,10,12,13,35,36.

Similar to the case of sense-codon recognition by tRNA, three universally conserved DC residues, A1492, A1493, and G530 of the ribosome’s 16S rRNA undergo key structural rearrangements during translation termination. In the RF-lacking termination complex, A1492 of helix 44 in 16S rRNA stacks with A1913 of H69. A1493 is flipped out and stabilizes the first two bases in the A-site stop codon. G530 stacks with the third base A in the stop codon. In the presence of RF, whether compact or extended, A1492 is flipped out towards G530 and interacts with the first two stop-codon bases. A1493 stacks with A1913, which is in close contact with A1492 in the RF-lacking termination complex. G530 stacks with the third stop-codon base (Fig. 3a, b).

Interaction of RF1 with the ribosomal decoding center. a, b Structures of the ribosomal decoding center in pre-accommodated ribosome complex (a) and accommodated ribosome complex (b). Red: compact RF1; dark blue: extended RF1. c, d Conformations of switch loop in pre-accommodated ribosome complex (c) and accommodated ribosome complex (d). Gold: A1492 and A1493; and orange: S12

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The switch loop, which was previously proposed to be involved in inducing a conformational change of RF110,36, shows no interaction with protein S12 or 16S rRNA in the compact form of RF1 (Fig. 3c) whereas in the extended form of RF1, the rearranged conformation of the switch loop is stabilized by interactions within a pocket formed by protein S12, the loop of 16S rRNA, the β-sheet of domain II, and A1493 and A1913 (Fig. 3b, d). Shortening the switch loop (302–304) resulted in a substantially slower, rate-limiting step in peptide release37, which indicates that the switch loop plays a role in triggering the conformational change of RF1.

Similar experiments were carried out for RF2 at 24 ms, 60 ms and 5 h reaction times. RF2 undergoes a conformational change from compact to expanded form similar to that of RF1 (Fig. 1e). As in the case of RF1, the switch loop of RF2 makes no contact with protein S12 or 16S rRNA. In the extended form of RF2, A1492 is flipped out from helix 44 (h44) of 16S rRNA and stacks on the conserved Trp319 of the switch loop, stabilizing the extended conformation of RF2 on the ribosome.

The ribosome complexes with RF1/2 bound in compact conformation seen here are distinct from those reported for the ribosome rescue complex, in which ArfA is bound in the A site lacking a stop codon14,15. In our structures, the conformation of the conserved 16S rRNA residues in the DC (A1492, A1493, and G530) is similar to the classical termination configuration10. In contrast, in the presence of ArfA, these residues adopt conformations known from sense-codon recognition14,15. It suggests that the compact RFs bind to the A site regardless of the conformation of the DC. The conformational change of RFs is likely due to the changes in the switch loop triggered by its interaction with protein S12 and 16S rRNA. This interaction is disrupted by the mutation A18T of ArfA, hence leaving RFs in the compact conformation15.

Our ribosome complexes with RF1/2 are also distinct from a recent ribosome complex with compact RF1, reported by Svidritskiy and Korostelev6. Shortening of the switch loop, combined with the addition of the antibiotic BlaS which prevents the GGQ motif from reaching the PTC, stabilizes ribosome-bound RF1 in a compact conformation6, distinct from the transient, compact RF1-structure observed here. In our structure, the SCR between the β4–β5 strands on domain II are bound loosely to the A site (Fig. 3a). In the BlaS-halted compact RF1 structure6, in contrast, the stop codon-recognition motif of RF1 has moved further into the A site by 5 Å, to a position almost identical to that of the fully accommodated, extended structure of RF1. The functional role of their structure is not immediately obvious, but if it can be interpreted as an authentic transition state analogue, the roles of our respective complexes would be complementary. We previously found that high accuracy of stop signal recognition depends on smaller dissociation constant (Km-effect) and larger catalytic rate constant (kcat-effect) for class-1 RF reading of cognate stop codons compared to near-cognate sense codons38. The Km-effect contributes by factors from 100 to 3000 and the kcat-effect by factors from 2 to 3000 to the overall termination accuracy values in the 103–106 range38. Accordingly, the present structure may represent binding of RFs in a transient state where rapid and codon-selective dissociation rates are responsible for the accuracy factor due to the Km-effect. Furthermore, Korostelev's structure6, with its comparatively deep interaction between the cognate stop codon and SCR center, could mimic the authentic transition state on the path from compact to the extended form of the RF. Accordingly, Korostelev's structure may illustrate additional selectivity due to the kcat-effect. To test these hypotheses, molecular computations28 based on our respective RF structures could be used to compare their stop codon selectivities with those of RFs in the post-termination state of the ribosome20.

In a recent paper on the role of RF3 in the dissociation of the release factors RF1 and RF239, the authors observed an interaction between domain I of RF1 and L7/L12 proteins, which assists the binding of RF1, as supported by complementary functional analysis using L7/L12 deletion mutants. However, such an interaction is not observed in our structures. Another recently published study using smFRET40 reported two states of the termination complex, non-rotated and rotated, in apparent contradiction to our results as we only found one, non-rotated state. The rotated-state subpopulation observed by Adio et al.40 may represent the post-termination ribosome unbound to RF1/RF2, as also suggested by previous single-molecule work from Puglisi and Gonzalez labs41,42.

Discussion

During translation termination, the release of the nascent peptide must be strictly coordinated with the recognition of a stop codon at the A site. Our cryo-EM analysis shows that in the presence of a class-1 RF the bacterial ribosome adopts several states. After rapid addition of RF1 or RF2 to a ribosomal termination complex with tripeptidyl-tRNA attached at the P site, we first observe the pre-accommodated RF-ribosome complex (compact form of RF) at 24 ms with the peptide attached to the P-site tRNA. This, we suggest, is the first step in the termination reaction. Second, at 60 ms, we observe the accommodated RF-ribosome complex with the extended form of RF and the tripeptide in the exit tunnel. Third, at a much later time point, we observe the post-accommodated RF-ribosome complex, with the extended form of RF without tripeptide in the exit tunnel (Supplementary Fig. 5). These pieces of evidence from our time-resolved experiments clearly reflect the sequence of events in termination of bacterial protein synthesis. A structure-based model for the stepwise interaction between ribosome and RF and the release of the nascent peptide from the termination complex during the translation termination process is presented in Fig. 4. It shows how the ribosome traverses (1) the pre-termination state with the stop codon at the A site, (2) the initial binding state (RF compact; pre-accommodated RF-ribosome complex), (3) the open catalytic state (RF open/extended; accommodated RF-ribosome complex) and (4) the state after peptide release. We suggest that the selective advantage of the compact RF-form is that it allows for rapid factor binding into and dissociation from an accuracy maximizing pre-accommodation state.

Structure-based model. The sequence of states is (1) the termination complex with the stop codon at the A site, (2) the initial binding state (RF compact; “pre-accommodated RF-ribosome complex”), (3) the open catalytic state (RF open/extended; “accommodated RF-ribosome complex”) and (4) the state after peptide release. (The later time point is not known from our experiment, and we only know from another experiment that the final state was seen after 45 s.) Blue: 50S large subunit; orange: 30S small subunit; green: tripeptide; brown: P-tRNA; pink: mRNA; red: compact RF; and blue-purple: extended RF

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Methods

Components for in vitro translation and fast kinetics

Buffers and all Escherichia coli (E. coli) components for cell-free protein synthesis were prepared as described22. Ribosomal release complexes (RC) contained tritium (3H) labeled fMet-Phe-Phe-tRNAPhe in the P site and had UAA stop-codon programmed A site. The mRNA sequence used to synthesize the peptide was GGGAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUUAAAUGUUCUUCUAAUGCAGAAAAAAAAAAAAAAAAAAAAA (ORF underlined and bold, SD underlined). Class-1 release factors (RFs), overexpressed in E. coli, had mainly unmethylated glutamine (Q) in the GGQ motif and the RF2 variant contained Ala in position 246. Rate constants for conformational changes of RFs in response to cognate A-site stop codon (kconf) and for ester bond hydrolysis (khydr) at different OH concentrations were estimated as described22. In short, purified release complexes (0.02 µM final concentration) were reacted at 25 °C with saturating amounts of RFs (0.8 µM final) in a quench-flow instrument, and the reaction stopped at different time points by quenching with 17% (final concentration) formic acid. Precipitated [3H]fMet-Phe-Phe-tRNAPhe was separated from the soluble [3H]fMet-Phe-Phe peptide by centrifugation. The amounts of tRNA-bound and free peptides were quantified by scintillation counting of the 3H radiation. Reaction buffer was polymix-HEPES with free Mg2+ concentration adjusted from 5 to 2.5 mM by addition of 2.5 mM Mg2+-chelating UTP. The rate constants for RF association to the A site at 25 °C, ka25, were estimated from their previously published values at 37 °C, ka37 = 60 µM−1 s−1 for RF1 and 23 µM−1 s−1 for RF238 through ka25 = (T2525)·(ŋ37/T37), where T is the absolute temperature and ŋ the water viscosity. Kinetics simulations were carried out with the termination reaction steps modeled as consecutive first-order reactions43.

Preparation of EM grids and time-resolved cryo-EM

Quantifoil R1.2/1.3 grids with a 300 mesh size were subjected to glow discharge in H2 and O2 for 25 s using a Solarus 950 plasma cleaning system (Gatan, Pleasanton, CA) set to a power of 10 W. Release complexes and RFs were prepared in the same way as for quench-flow experiments, except the release complexes were unlabeled. For each time point (24 ms and 60 ms), 4 µM of release complexes in polymix-HEPES with 2.5 mM UTP and 6 µM of class-1 release factors in the same buffer were injected into the corresponding microfluidic chip at a rate of 3 µl/s such that they could be mixed and sprayed onto a glow-discharged grid as previously described33. The final concentration of the release complexes and the class-1 release factors after rapid mixing in our microfluidic chip was 2 µM and 3 µM, respectively. As the mixture was sprayed onto the grid, the grid was plunge-frozen in liquid ethane-propane mixture (37%:63%) and stored in liquid nitrogen until it was ready to be imaged.

Preparation of EM grids and blotting-plunging cryo-EM

Grids of RC0 and long-incubation complex were prepared with the following protocol. 3 uL sample was applied in the holey grids (gold grids R0.6/1 300 mesh, which was plasma cleaned using the Solarus 950 advanced plasma cleaning system (Gatan, Pleasanton, CA) for 25 s at 10 W using hydrogen and oxygen plasma). Vitrification of samples was performed in a Vitrobot Mark IV (FEI company) at 4 °C and 100% relative humidity by blotting the grids once for 6 s with a blot force 3 before plunging them into the liquid ethane-propane mixture.

Cryo-EM data collection

Time-resolved cryo-EM grids were imaged either with a 300 kV Tecnai Polara F30 TEM or a Titan Krios TEM. The images were recorded at a defocus range of −1 to −3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.66 Å or 1.05 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image. Blotting-plunging cryo-EM grids were imaged with a 300 kV Tecnai Polara F30 TEM. The images were recorded at a defocus range of 1–3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.24 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image.

Cryo-EM data processing

The beam-induced motion of the sample and the instability of the stage due to thermal drift was corrected using the MotionCor2 software program44. The contrast transfer function (CTF) of each micrograph was estimated using the CTFFIND4 software program45. Imaged particles were picked using the Autopicker algorithm included in the RELION 2.0 software program46. For each time point (Supplementary Figs 6 and  7), 2D classification of the recorded images were used to separate 70S ribosome-like particles from ice-like and/or debris-like particles picked by the Autopicker algorithm and to classify the particles that were picked for further analysis into 70S ribosome-like particle classes. These particle classes were then combined into a single dataset of 70S ribosome-like particles and subjected to a round of 3D classification for the purpose of eliminating those obvious contaminants from the rest of the dataset. This classification was set for 10 classes with the following sampling parameters: Angular sampling interval of 15°, offset search range of 5 pixels and offset search step of 1 pixel. The sampling parameters were progressively narrowed in the course of the 50 classification iterations, down to 3.7° for the angular sampling interval. At the end of the first classification round, two classes were found inconsistent with the known structure of the 70S ribosomes and were thus rejected. The rest of the particles were regrouped together as one class. All particles from this class were re-extracted using unbinned images. A consensus refinement was calculated using these particles. The A site of the 70S ribosome displays fractioned density indicating heterogeneity, then, therefore, the signal subtraction approach was applied. The A-site density was segmented out of the ribosome using Segger in Chimera47. The mass of density identified as release factor was used for creating a mask in RELION with 3 pixels extension and 6 pixels soft edge using relion_mask_create. This mask was used for subtracting the release factor-like signal from the experimental particles. The new particles images were used directly as input in the masked classification run with the number of particles set for ten classes, and with the mask around the release factor-binding region. This run of focused classification resulted in two separate classes, one with compact and one with extended conformation of the release factors. The corresponding raw particles were finally used to calculated consensus refinements. The local resolution of the final maps was computed using ResMap48.

For the RC0 complex dataset, the software MotionCor244 was used for motion correction and dose weighting. Gctf49 was used for estimation of the contrast transfer function parameters of each micrograph. RELION46 was used for all other image processing steps. Particles picking was done automatically in RELION. Boxed out particles were extracted from dose-weighted micrographs with eight times binning. 2D classifications were initially performed on bin8 particle stacks to remove false positive particles from the particle picking step. 3D classification were performed on bin4 particle stacks. Classes from bin4 and bin2 3D classification showing high-resolution features were saved for further processing steps. Un-binned particles from this class were re-extracted and subjected to auto-refinement. The final density map was sharpened by applying a negative B-factor estimated by automated procedures. Local resolution variations were estimated using ResMap48 and visualized with UCSF Chimera47.

Model building and refinement

Models of the E. coli 70S ribosome (5MDV, 5MDW, and 5DFE) were docked into the maps using UCSF Chimera. The pixel size was calibrated by creating the density map from the atomic model and changing the pixel size of the map to maximize the cross-correlation value. For the compact RF1 model, a homology model was generated with the crystal structure of the RF1 (PDB ID: 1ZBT) as a template using the SWISS-MODEL online server50. This homology model was rigid-body-fitted into the map using UCSF Chimera, followed by manual adjustment in Coot51. Due to the lack of density, domain I of RF1 was not modeled.

Figure preparation

All figures showing electron densities and atomic models were generated using UCSF Chimera47.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The atomic coordinates and the associated maps have been deposited in the PDB and EMDB with the accession codes 20173, 20184, 20187, 20188, 20193, 20204, 6ORE, 6ORL, 6OSQ, 6OST, 6OT3, and 6OUO. The source data underlying Supplementary Figs 2 and 3 are provided as a Source Data file.

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Acknowledgements

This work was supported by HHMI and grants NIH R01 GM55440 and R01 GM29169 (to J.F.), the Swedish Research Council, and the Knut and Alice Wallenberg Foundation (to M.E.), and the Sederholms travel stipend (Uppsala University) (to G.I.).

Author information

Author notes
  1. These authors contributed equally: Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar.

Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032, USA

    Ziao Fu, Sandip Kaledhonkar, Robert A. Grassucci & Joachim Frank

  2. Department of Cell and Molecular Biology, Uppsala University, Uppsala, 751 24, Sweden

    Gabriele Indrisiunaite & Måns Ehrenberg

  3. Department of Biological Sciences, Barnard College, New York, NY, 10027, USA

    Binita Shah

  4. Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

    Ming Sun, Bo Chen & Joachim Frank

Contributions

Z.F., G.I., S.K., B.S., M.S., B.C., R.A.G., M.E., and J.F. conceived and designed experiments. G.I. carried out biochemical experiments. Z.F., S.K., G.I., and B.S. performed time-resolved cryo-EM experiments. Z.F., S.K., G.I., B.S., and M.S. performed image processing and atomic modeling. Z.F., S.K., G.I., and B.S. analyzed the data. Z.F., G.I., S.K., M.E., and J.F. wrote the manuscript.

Corresponding author

Correspondence to Joachim Frank.

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Abstract

The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore-forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Examination of additional point mutants unexpectedly revealed that the CTS of Bim directly interacts with Bax, is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.

Introduction

Apoptosis is a highly conserved form of programmed cell death that can be triggered by extrinsic or intrinsic signals. It plays a fundamental role in maintaining homeostasis by eliminating old, excessive or dysfunctional cells in multicellular organisms (Kerr et al., 1972). Defective regulation of apoptosis has been found in many diseases (Favaloro et al., 2012) and is considered one of the hallmarks of cancer (Hanahan and Weinberg, 2011).

Bcl-2 family proteins play a decisive role in apoptosis initiated by intrinsic signaling by regulating the integrity of the mitochondrial outer membrane (MOM). Commitment to apoptosis is generally regarded as due to MOM permeabilization (MOMP) releasing cytochrome c and pro-apoptotic factors from the intermembrane space into the cytoplasm. These factors activate the executioner caspases that mediate cell death (Chipuk et al., 2006). Direct interactions between Bcl-2 family proteins govern both initiation and inhibition of MOMP (Kale and Osterlund, 2017). The Bcl-2 family of proteins that regulate apoptosis includes the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1 that inhibit the process and share four Bcl-2 homology domains. These homology domains, referred to as BH domains, are also shared by the pro-apoptotic proteins Bax and Bak that permeabilize the MOM directly. Both pro- and anti-apoptotic multi-domain Bcl-2 family proteins are regulated by direct binding interactions with a group of proteins including Bim, Bid, Puma, Hrk, Bad and Noxa that contain a single region of homology, the Bcl-2 homology domain number 3, and are therefore referred to collectively as BH3-proteins. These proteins promote apoptosis by releasing sequestered activated Bax, Bak and BH3-proteins that activate Bax and Bak from one or more of the anti-apoptotic proteins. The subset of BH3 proteins that bind to and activate Bax or Bak include Bid, Bim and Puma (Chi et al., 2014). Thus far, the biochemical basis for the differences between BH3-proteins that inhibit anti-apoptotic proteins and those that activate Bax and Bak has been attributed entirely to differences in affinities of the BH3-domain for the BH3-peptide binding sites on multi-domain pro- and anti-apoptotic proteins. However, static affinities and variations in expression levels permit only coarse regulation of cell death. Changes in the equilibrium binding of Bcl-2 family proteins on the MOM enable finer control. For example, at physiologic concentrations, the BH3-protein Bid only activates Bax after Bid has bound to a membrane and undergone a specific conformational change (Lovell et al., 2008; Shamas-Din et al., 2013a). Binding to membranes also enables interaction of Bid with MTCH2 on the MOM to greatly accelerate the Bid conformational change that results in Bax activation (Shamas-Din et al., 2013a). However, it remains unclear whether membrane interactions by other BH3-proteins like Bim contribute to Bax activation.

The BH3-protein Bim is an important mediator of apoptosis initiated by many intracellular stressors (Concannon et al., 2010; Mahajan et al., 2014; Puthalakath et al., 2007). Three major isoforms of Bim result from alternative mRNA splicing: BimEL, BimL, and BimS (O'Connor et al., 1998). All three isoforms include the BH3-domain required for binding other Bcl-2 family proteins, and a C-terminal sequence (CTS) that binds the protein to the MOM (Wilfling et al., 2012). BimEL and BimL also share a dynein light chain binding motif (LC1) that sequesters these isoforms at the cytoskeleton (Lei and Davis, 2003). However, recent evidence also suggests that the LC1 sequence mediates Bim oligomerization by binding to the DLC1 protein (Singh et al., 2017). Isoform BimS does not contain the LC1-binding motif (Lei and Davis, 2003), and is rarely present in healthy cells, while BimL and BimEL are present in most tissue types (O'Reilly et al., 2000). Bim has a particularly important function as a regulator of anti-apoptotic proteins, as it binds and thereby inhibits by mutual sequestration all known anti-apoptotic proteins (Chen et al., 2005; Shamas-Din et al., 2013b). Until recently, it was unknown why Bim binds to Bcl-XL with sufficient affinity to resist displacement by small molecule BH3-mimetics, while other BH3-proteins, such as Bad, are displaced (Aranovich et al., 2012). In addition to interactions via the BH3-domain, residues within the Bim CTS bind to Bcl-XL, and thereby increase the affinity of the interaction by ‘double-bolt locking’ providing an explanation for the observations with BH3 mimetic drugs (Liu et al., 2019). Here, we investigated whether the CTS of Bim also contributes to the functional and physical interactions between Bim and Bax.

We demonstrate that both primary cells and cell lines have a range of apoptotic responses to the expression of a truncated BimL protein lacking the CTS (BimL-dCTS), while expression of full-length BimL was sufficient to kill all of these cells. To determine the molecular mechanism that underlies this difference, the two pro-apoptotic functions of Bim; activation of Bax and inhibition of Bcl-XL, were quantified using purified full-length BimL and BimL mutant proteins and cell-free assays. Replacing the CTS of Bim with an alternative tail-anchor that binds the protein to mitochondrial membranes did not fully restore Bax activation function, demonstrating that sequences within the Bim CTS rather than membrane binding contribute to Bax activation. Site-directed mutagenesis of the Bim CTS also revealed residues important for binding to membranes that were not required for Bax activation (e.g. I125). Furthermore, specific residues within the CTS were identified that are required for BimL to efficiently activate Bax, but that are not required for BimL to bind to and inhibit Bcl-XL. Evidence in cell free assays demonstrated that BimL CTS residues L129 and I132 physically interact with Bax in the BH3 binding groove and are required for Bim to activate Bax. Together, our data demonstrates that the unusual sequence of the CTS of Bim separately controls both membrane binding and Bax activation.

Results

The CTS of Bim variably contributes to the pro-apoptotic activity of Bim in different cell lines

Removing the CTS from Bim abrogates pro-apoptotic activity in HEK293 cells (Weber et al., 2007). While this observation has generally been ascribed to loss of binding of Bim to MOM our observation that the CTS is also involved in binding BimEL to Bcl-XL (Liu et al., 2019) suggested that there may be other explanations for the loss of pro-apoptotic activity for Bim when the CTS is removed. To determine the contribution of the Bim CTS to pro-apoptotic activity, a BimL mutant was generated in which the previously characterized membrane binding domain (carboxyl-terminal residues P121- H140) were deleted (BimL-dCTS) (Wilfling et al., 2012; Liu et al., 2019). This mutant was expressed in cells and the effectiveness of induction of cell death was compared to expression of full-length BimL by confocal microscopy. To detect expression of the constructs in live cells, they included an N-terminally fused Venus fluorescent protein (indicated by a superscripted v in the name). Thus, a construct in which Venus was fused to the amino-terminus of BimL is referred to here as VBimL while the mutant lacking the CTS is VBimL-dCTS. As an inactive control, we used VBimL-4E a mutant in which four conserved hydrophobic residues in the BH3-domain of BimL were replaced with glutamate, thereby preventing binding to all other multi-BH domain Bcl-2 family proteins (Chen et al., 2005; Liu et al., 2019).

To assay pro-apoptotic activity, the constructs were expressed in primary cells and cell lines and both expression and cell death were measured using confocal microscopy. Apoptosis was assessed by detecting externalization of phosphatidylserine by Annexin V staining in cells expressing detectable levels of VBimL or the VBimL mutants as measured by Venus fluorescence. As a positive control for activation of Bax VtBid, the activated form of the BH3-protein Bid fused to the C-terminus of the Venus fluorescent protein was also expressed in cells. As expected, expression of VBimL induced apoptosis in all cell types tested, while the negative control protein VBimL-4E did not (Figure 1A). Similarly, VtBid induced apoptosis in all the cell types except HEK293 cells. As reported previously for Bim-dCTS, the fluorescent version (VBimL-dCTS) failed to induce cell death in HEK293 cells (Weber et al., 2007). In contrast, expression of VBimL-dCTS induced apoptosis to levels similar to VBimL in HCT116, BMK and MEF cells but may have reduced potency in CAMA-1 cells. Comparing the AnnexinV intensities for individual cells at a variety of equivalent expression levels of the Bim mutants across the different cell types revealed that the CTS of Bim was required for the pro-apoptotic activity of Bim in HEK293 cells (Figure 1—figure supplement 1).

To determine if this difference in response to VBimL-dCTS expression is a function of the extent to which the apoptotic machinery is loaded in MOM, mitochondria were purified from cells resistant (HEK293) and sensitive (MEF) to VBimL-dCTS expression and assayed by BH3-profiling (Potter and Letai, 2016). This assay measures loading of anti-apoptotic proteins with BH3-proteins or active Bax/Bak. Unlike BH3-profiling experiments conducted with BH3-peptides, in these experiments purified full-length proteins were used. Thus, purified cBid, BimL, BimL-dCTS, Bad and Noxa proteins were incubated with mitochondria from each of the cell lines and MOMP was measured by separating supernatant and pellet fractions for each reaction, and immunoblotting for cytochrome c released from the intermembrane space as previously described (Pogmore et al., 2016). Immunoblots were quantified and MOMP assessed as % cytochrome c released (Figure 1B). As expected from the data in Figure 1A, addition of recombinant BimL was sufficient to induce cytochrome c release from mitochondria from both HEK293 and MEF cells. However, addition of BimL-dCTS induced cytochrome c release only in the MEF mitochondria confirming that resistance to BimL-dCTS in HEK293 cells is manifest at mitochondria. Mitochondria purified from HEK293 cells were more sensitive to BimL protein than to recombinant cBid, a phenomenon that is also seen when VtBid is expressed in these cells (Figure 1A). This result may be due to the inherent differences between Bim and Bid reported previously (Sarosiek et al., 2013). Nevertheless, in HEK293 cells, VtBid was more active than BimL-dCTS and functionally equivalent to VBimL in every other cell line tested.

One potential explanation for the difference in response to BimL-dCTS and BimL is that the mitochondria in the cell lines have different dependencies on multi-domain anti-apoptotic proteins for survival, a phenomenon known as priming. If BimL-dCTS has lost one of the functions of Bim such as activating Bax or Bak or inhibiting one of the multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 it would be expected to have different activities on mitochondria with different priming. Therefore, to better understand why BimL-dCTS can only permeabilize MEF mitochondria and not mitochondria from HEK293 cells, we compared the sensitivity of mitochondria from the two cell types to addition of BH3-proteins Bad or Noxa that inhibits Bcl-2 and Bcl-XL or Mcl-1, respectively, but that do not activate Bax or Bak (Kale and Osterlund, 2017). Incubation of full-length Bad and/or Noxa with mitochondria from HEK293 cells failed to induce cytochrome c release, while the addition of Noxa or Bad was sufficient to permeabilize MEF mitochondria (Figure 1B). This data suggests that HEK293 cells not depend on expression of Bcl-2, Bcl-XL or Mcl-1 sequestering active Bax, Bak or their BH3-activators while mitochondria from MEFs depend on expression of Mcl-1 and Bcl-XL to prevent apoptosis (Lessene et al., 2013). The results further suggest that removal of the CTS from BimL results in a mutant protein that only kills cells dependent on one or more multi-domain anti-apoptotic proteins for survival. That BimL-dCTS does not kill HEK293 cells further suggests that it does not activate sufficient Bax or Bak to overcome the unoccupied anti-apoptotic proteins in this cell line. In this way, BimL-dCTS functions as a sensitizer similar to proteins like Bad and Noxa. However, unlike other relatively specific sensitizer proteins, the BH3-region of BimL-dCTS binds to and inhibits Bcl-2, Bcl-XL and Mcl-1. Indeed, we have shown that in live cells BimEL-dCTS binds to and inhibits Bcl-2 and Bcl-XL but is more easily displaced than BimEL by small molecule BH3 mimetics (Liu et al., 2019).

Full-length BimL is required to kill cultures of primary cortical neurons

Our data with cell lines and their respective purified mitochondria suggests that BimL-dCTS does not kill cells that do not depend on anti-apoptotic proteins for survival. To test this in a more biologically relevant system, we cultured primary murine cortical neurons and assayed their response to expression of the BimL mutants. To enable regulated expression in primary cortical neurons the coding regions for VBimL, VBimL-4E, and VBimL-dCTS were cloned into a tetracycline-responsive lentiviral vector, and introduced into primary cortical neuron cultures through lentiviral infection. After culture for 16 days in vitro, BimL expression was induced in the neurons by the addition of doxycycline. Neuronal cell death was assayed using confocal microscopy after staining neurons with TMRE, a dye that only accumulates in active mitochondria. Thus, a lack of TMRE dye accumulation (TMRE negative) indicates loss of mitochondrial transmembrane potential and in response to expression of a BH3-protein is an early indication of commitment to cell death. Quantification of Venus-expressing neuronal cell bodies revealed that as expected VtBid and VBimL expression killed cultured primary neurons while VBimL-4E did not (Figure 2A–B). However, the expression of VBimL-dCTS was largely ineffective to induce cell death in cultured primary cortical neurons (Figure 2B). Our data is consistent with previous reports suggesting that primary murine cultures of hippocampal neurons become resistant to induction of apoptosis by external stimuli over time in culture. This resistance has been reported to be due to a difference in Bcl-2 family protein expression that results in decreased mitochondrial ‘priming’, explaining why our cultures of primary cortical neurons are resistant to VBimL-dCTS (Sarosiek et al., 2017).

To determine if resistance to induction of cell death by BimL-dCTS is due to differential sensitivity of neuronal mitochondria to induction of MOMP by BimL and BimL-dCTS, mitochondria were isolated from embryonic day 15 (E15) mouse brains, the same age used to culture primary cortical neurons. Brain mitochondria were used instead of isolating mitochondria from neuronal cultures due to the low yield from primary cultured neurons. Untreated mitochondria from day E15 brain released only low levels of cytochrome c. As expected, addition of 0.1 nM recombinant BimL was sufficient to elicit MOMP as measured by cytochrome c release and detection in the supernatant. In contrast, 100 times more BimL-dCTS (10 nM) failed to induce MOMP (Figure 2C).

Taken together our data suggest that BimL-dCTS kills cells in which the mitochondria are sensitive to inhibition of anti-apoptotic proteins by sensitizers such as Bad and Noxa. Thus, BimL-dCTS did not permeabilize mitochondria extracted from HEK293 cells or E15 whole murine brains, and as a result, BimL-dCTS expression did not kill HEK293 cells or primary cultures of cortical neurons. This finding suggests that inhibition of anti-apoptotic proteins is not sufficient to kill these cells. Therefore, BimL-dCTS differs mechanistically from BimL as the latter kills both cell types resistant and sensitive to BimL-dCTS. Compared to BimL, BimL-dCTS is missing the membrane-binding domain and therefore is not expected to localize at mitochondria (Liu et al., 2019); however, the relationship between Bim binding to membranes and Bim-mediated Bax activation has not been extensively studied. To determine how the molecular mechanism of BimL-dCTS differs from BimL the activities of the proteins were analyzed using cell-free assays.

The Bim CTS mediates BimL binding to both Bax and membranes

To investigate the pro-apoptotic mechanism of BimL and BimL-dCTS without interference from other cellular components, both were purified as full-length recombinant proteins and assayed using liposomes and/or isolated mitochondria. To measure direct-activation of Bax by Bim, either BimL or BimL-dCTS was incubated with recombinant Bax and liposomes encapsulating the dye and quencher pair: ANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt) and DPX (p-Xylene-Bis-Pyridinium Bromide). In this well-established assay (Chi et al., 2014), increasing amounts of BimL activated Bax results in membrane permeabilization measured as an increase in fluorescence due to the release and separation of encapsulated dye and quencher (Figure 3A). This result is consistent with previous observations that picomolar concentrations of BimL induce Bax-mediated membrane permeabilization (Sarosiek et al., 2013). In contrast, three orders of magnitude higher concentrations of BimL-dCTS were required to induce Bax-mediated liposome permeabilization (Figure 3A), suggesting that either or both of binding to membranes and the specific CTS of Bim are required for efficient Bax activation. As expected, similar results were obtained for Bax-mediated release of mitochondrial intermembrane space proteins (Figure 3B). For these experiments, MOMP was measured as release of the fluorescent protein mCherry fused to the N-terminal mitochondrial import signal of SMAC (SMAC-mCherry) from the intermembrane space of mitochondria (Shamas-Din et al., 2014). Similar to the results with liposomes (Figure 3A), and mitochondria from cell lines (Figures 1–2) BimL but not BimL-dCTS triggered Bax mediated SMAC-mCherry release from mitochondria isolated from Bax - /- Bak-/-cells (Figure 3B). In experiments with liposomes and mitochondria, very small amounts of Bim were sufficient to trigger membrane permeabilization because once activated, Bax recruits and activates additional Bax molecules (Tan et al., 2006). To assess the impact of the Bim CTS on the interaction between Bim and Bax, binding was measured using Förster resonance energy transfer (FRET). For these experiments, recombinant BimL proteins were labeled with the donor fluorophore Alexa568, while Bax was labeled with the acceptor fluorophore Alexa647. Unexpectedly, and unlike the BH3-only protein tBid (Lovell et al., 2008), BimL bound to Bax even in the absence of membranes (Figure 3C), while BimL-dCTS had no relevant Bax binding in the presence or absence of mitochondrial-like liposomes (Figure 3C–D). Binding of Bim to Bax in solution suggests that the CTS of Bim may be directly involved in Bim-Bax heterodimerization independent of Bim binding to membranes.

To confirm in our system that the labeled BimL proteins bind to membranes via the CTS sequence, binding of Alexa568-labeled recombinant BimL and BimL-dCTS to DiD labeled liposomes was measured by FRET (Figure 4A). In these experiments, DiD serves as an acceptor for energy transfer from Alexa568-labeled BimL. The same approach was used to quantify BimL binding to mitochondrial outer membranes with mitochondria isolated from BAK-/-mouse liver (Figure 4B), which lack Bax and Bak (Shamas-Din et al., 2013a). In both cases, BimL spontaneously bound to membranes with picomolar affinity, while stable binding of BimL-dCTS to liposomes and mitochondria was not-detectable (Figure 4A–B). Furthermore, BimL-dCTS again had no relevant binding to Bax even in the presence of purified mitochondria (Figure 4C).

Taken together, our data strongly suggest that the CTS of Bim is required for both BimL to bind to membranes in vitro and for binding Bax with or without membranes. Alternatively purified BimL-dCTS may be completely non-functional. To demonstrate that purified BimL-dCTS binds to and inhibits Bcl-XL as shown for VBimL-dCTS expressed in cells (Figure 1) and in Liu et al. (2019), inhibition of Bcl-XL was measured using liposomes and mitochondria.

The CTS is not required for BimL to inhibit Bcl-XL

In addition to direct Bax activation, Bim promotes apoptosis by binding to Bcl-XL and displacing either activator BH3-proteins (Mode 1) or activated Bax or Bak (Mode 2) (Llambi et al., 2011). In the ANTS/DPX liposome dye release assay, BimL-dCTS was functionally comparable to the well-established Bcl-XL inhibitory BH3-protein Bad in reversing Bcl-XL-mediated inhibition of cBid (Figure 5A) or Bax (Figure 5B). Consistent with the observation that BimL-dCTS was less resistant to displacement by BH3 mimetics in live cells, in cell-free assays BimL-dCTS was also less effective than BimL at displacing cBid or Bax from Bcl-XL (Liu et al., 2019). Nevertheless, when assayed with mitochondria BimL-dCTS disrupted the interaction between tBid and Bcl-XL resulting in Bax activation and permeabilization of mitochondria as measured by cytochrome c release (Figure 5C, solid black line). This activity is due to inhibition of Bcl-XL function, as in controls without Bcl-XL the same concentration of BimL-dCTS did not directly activate sufficient Bax to mediate MOMP (Figure 5C, dashed black line). Thus, purified BimL-dCTS is functional and can initiate MOMP by displacing direct-activators (Mode 1) or activated Bax (Mode 2) from Bcl-XL (Figure 5A–C). Finally, BimL-dCTS labeled with Alexa568 retained high-affinity binding for Bcl-XL labeled with Alexa647 both in solution (Kd <16 nM) and on membranes (~35 nM apparent Kd on liposomes and on mitochondria) as measured by FRET (Figure 6B).

Different residues in the Bim CTS regulate membrane binding and Bax activation

To identify which residues in the Bim CTS mediate binding to membranes and/or Bax we generated a series of point mutations. Sequence analysis using HeliQuest software (Gautier et al., 2008) predicts that the Bim CTS forms an amphipathic α-helix (Figure 6A). Two arginine residues (R130 and 134) are predicted to be on the same hydrophilic side of the helix, whereas hydrophobic residues (e.g. I125, L129, I132) face the other side (Figure 6A). To determine the functional importance of these residues, Bim CTS mutants were created including: BimL-CTS2A in which R130 and R134 were mutated to alanine; and a series of single hydrophobic residue substitutions by glutamate (V124E, I125E, L129E, and I132E) (Figure 6A). To compare the effects of the CTS mutations on BimL-binding interactions and function, we measured by FRET the Kds for the various binding interactions and the activities of the mutants to promote Bax-mediated liposome permeabilization as EC50’s for ANTS release (Figure 6B and Figure 6—figure supplement 1A–E).

Mutation of individual hydrophobic residues on the hydrophobic side of the Bim CTS (BimL-I125E, BimL-L129E or BimL-I132E) abolished binding to membranes (Figure 6B). In contrast, mutations on the other side of the helix including BimL-V124E and BimL-CTS2A had less effect on Bim binding to membranes (Figure 6B). Despite the dramatic changes in affinity for membranes among Bim CTS mutants, the mutations did not abolish binding to Bax both in the presence and absence of membranes (Figure 6B). Indeed most of the mutants had Kd values for binding to Bax of less than 100 nM and to our surprise many of them bound to Bax better in solution compared to when membranes were present. The presented Kds under ‘membranes present’ is an estimation of the effective Kd (combined solution and two-dimensional Kds), as we are not able to precisely determine the quantity of protein-complexes that form in solution or on the membrane. Nevertheless, this data reflects the situation in cells and further confirms that binding to membranes and Bax are independent functions of the Bim CTS . In the case of BimL-I125E, a mutant that activates Bax to permeabilize liposomes, the initial interaction with Bax must occur in solution as neither protein spontaneously binds to membranes (Figure 6B).

Unexpectedly, there was not a good correlation between BimL binding to membranes and Bax activation. For example, while BimL bound to membranes with a Kd of 31 pM, BimL-CTS2A and BimL-I125E bound to membranes with Kds of ~600 and>1000 pM, respectively yet both mutants triggered Bax-mediated membrane permeabilization, demonstrating that specific residues in the CTS rather than binding to membranes enabled BimL to mediate Bax activation. Moreover, BimL binding to Bax was also not sufficient to activate Bax efficiently. BimL-L129E and BimL-I132E are two Bim mutants that do not bind membranes, retain reasonable affinities for Bax in the presence of membranes (Kds ~ 100–200 nM), but were unable to activate Bax (Figure 6B). These results indicate that these two residues play a key function in Bax activation. As expected, the negative control BimL-4E mutant does not bind to nor activate Bax even though its CTS is intact and the protein binds membranes (Figure 6B). This result confirms the essential role of the BH3-domain and suggests that the Bim CTS provides a secondary role in Bax binding rather than providing an independent high affinity binding site that is sufficient to activate Bax.

Both functional and binding assays for the various point mutants suggest that specific residues in the Bim CTS participate in Bim-Bax protein interactions that lead to Bax activation; however, these mutants did not clearly separate the membrane binding function of the CTS of Bim from a potential function in Bax activation. Thus, it remains possible that restoring membrane binding to BimL-dCTS would be sufficient to restore Bax activation function. To address this, we fused the mitochondrial tail-anchor from mono-amine oxidase (MAO residues 490–527, UniProt: P21397-1) to the C-terminus of BimL-dCTS to restore membrane binding with a sequence unlikely to contribute to Bax activation directly. This protein, BimL-dCTS-MAO, and BimL bound to mitochondrial-like liposomes and purified mitochondria (Figure 7A and Figure 7—figure supplement 1A respectively). As expected, a population of these recombinant Bim proteins remains in solution. To directly assess the Bim CTS contribution to the binding and activation of Bax on the membrane surface, we incubated a defined amount of recombinant Alexa568-labeled BimL or BimL-dCTS-MAO proteins with 0.5 mg/mL ANTS/DPX filled liposomes, then isolated the liposomes using size-exclusion chromatography. Using this procedure, we excluded all recombinant Bim that remained in solution, and obtained the membrane-bound BimL or BimL-dCTS-MAO at a concentration of ~5 nM as calculated based on Alexa568 fluorescence intensity (Precise concentrations labeled in Figure 7B). This equates to 0.6 Bim molecules per liposome. Addition of Bax to these liposomes resulted in ANTS/DPX release for roughly 60% of the liposomes in incubations containing BimL-membrane-bound liposomes, but no significant release from the liposomes in incubations containing BimL-dCTS-MAO-bound liposomes (Figure 7B), suggesting that the Bim CTS contributes to the activation of Bax even on the membrane surface.

To directly measure binding to Bax, Alexa568-labeled BimL and BimL-dCTS-MAO were incubated with increasing concentrations of Alexa647-labeled Bax protein. In the absence of membranes, no detectable FRET was measured between Alexa568-labeled BimL-dCTS-MAO with Alexa647-labeled Bax (Figure 7C), suggesting that the specific sequence of the Bim CTS contributes to the Bim-Bax interaction in solution. However, when Alexa568-labeled BimL-dCTS-MAO bound to the liposome membrane was isolated by size-exclusion chromatography as described above, a FRET signal was detected between it and Alexa647-labeled Bax. We also detected FRET between Alexa568-labeled BimL-dCTS-MAO and Alexa647-labeled Bax in the presence of purified mitochondria (Figure 7—figure supplement 1B). Although these incubations contained both soluble and membrane-bound Bim protein, we conclude the interaction occurred on the MOM as no FRET signal was detected between these proteins in solution (Figure 7C). Despite measureable binding between BimL-dCTS-MAO and Bax on the liposome membrane (Figure 7D), BimL retained a higher affinity than BimL-dCTS-MAO for Bax (Kds of 21 nM and 49.6 nM, respectively). Together these data suggest that the CTS of Bim contributes to both binding to (Figure 7D) and activation of Bax (Figure 7B) on the liposome membrane.

Residues within the Bim CTS are proximal to Bax in solution and on mitochondrial membranes

Our binding and mutagenesis data suggest that the Bim CTS binds to and activates Bax in solution and on membranes. To detect this binding interaction, we used a photocrosslinking approach, in which a BimL protein was synthesized with a photoreactive probe attached to a single lysine residue positioned in the CTS using an in vitro translation system containing 5-azido-2-nitrobenzoyl-labled Lys-tRNALys that incorporates the lysine analog (εANB-Lys) into the polypeptide when a lysine codon in the BimL mRNA is encountered by the ribosome. The BimL synthesized in vitro was also labeled by 35S via methionine residues enabling detection of BimL monomers and photoadducts by phosphor-imaging.

The radioactive, photoreactive BimL protein was incubated with a recombinant His6-tagged Bax protein in the presence of mitochondria isolated from BAK-/- mouse liver lacking endogenous Bax and Bak to prevent competition and increase BimL-Bax protein interactions. Mitochondrial proteins were then separated from the soluble ones by centrifugation. Both soluble and mitochondrial fractions were photolyzed to activate the ANB probe generating a nitrene that can react with atoms in close proximity (<12 Å from the Cα of the lysine residue). Thus, for photoadducts to form, the atoms of the bound Bax molecule are likely to be located in or near the binding site for the Bim CTS. The resulting photoadduct between the BimL and the His6-tagged Bax was enriched by Ni2+-chelating agarose resin and separated from the unreacted BimL and Bax monomers using SDS-PAGE. The 35S-labeled BimL in the photoadduct with His6-tagged Bax and BimL monomer bound to the Ni2+-beads specifically via the His6-tagged Bax or nonspecifically were detected by phosphor-imaging. A BimL-Bax-specific photoadduct was detected when the ANB probe was located at four different positions in the Bim CTS on both hydrophobic and hydrophilic surfaces of the potential α-helix (Figure 8A). These photoadducts have the expected molecular weight for the Bim-Bax dimer, and were not detected or greatly reduced when the ANB probe, the light, or the His6-tagged Bax protein was omitted (Figure 8A). Consistent with the BimL-Bax interaction detected by FRET in both solution and membranes, the BimL-Bax photocrosslinking occurred in both soluble and mitochondrial fractions. Less photocrosslinking occurred in the mitochondrial fraction likely due to the fact that in membranes homo-oligomerization of activated Bax competes with hetero-dimerization between BimL and Bax.

As expected, when the ANB probe was positioned in the Bim-BH3 domain as a positive control BimL-Bax photocrosslinking was detected in both soluble and mitochondrial fractions (Figure 8B). Crosslinking with the Bim BH3-domain is consistent with the BH3 interaction with the canonical groove or trigger pocket that is well supported by experimental evidence including co-crystal structures and NMR models (Gavathiotis et al., 2008; Robin et al., 2015). Furthermore, loss of photocrosslinking for BimL mutants with the BH3-4E mutation that abolished binding to Bax demonstrates that direct binding between the proteins is required for crosslinking to be detectable (Figure 8C). Therefore, the crosslinking data suggests that similar to the BH3-domain, the Bim CTS binds to Bax. To further demonstrate that the CTS of Bim binds to Bax independent of both membrane binding and Bax activation, the experiment was repeated with BimL-L129E, a mutant that binds Bax without activating it and that does not bind membranes (Figure 6B). As shown in Figure 8C, the L129E mutant photocrosslinked to Bax in both the soluble and mitochondrial fractions. Furthermore, this mutant also photocrosslinked to Bcl-XL (Figure 8C), consistent with data demonstrating that the Bim CTS also binds to this anti-apoptotic protein (Figure 6B and Liu et al., 2019). The qualitative photocrosslinking data (Figure 8) and the quantitative FRET data (Figure 6B) obtained from the BimL proteins with and without the CTS or BH3-domain mutation are consistent, and both support a model in which the CTS interacts with membranes and binds to Bax, thereby enhancing BH3-domain mediated Bax activation.

The Bim CTS binds to the BH3-binding pocket on Bax

To identify the binding site for the Bim CTS in Bax, we used a chemical crosslinking approach. Unlike the photocrosslinking approach that does not reveal the location of the binding site, the chemical crosslinker bismaleimidohexane (BMH) contains two sulfhydryl reactive moieties separated by a 13 Å spacer, and thus formation of a BMH-crosslinked Bim-Bax dimer requires a cysteine in Bim that is in close proximity with another cysteine in the interacting Bax. Therefore, a successful crosslink indicates a close proximity between the two cysteines, potentially revealing the Bim-binding site in Bax.

We used a structurally well-defined Bim-Bax interaction to validate this crosslinking approach. According to the crystal structure (PDB ID 4ZIE; Robin et al., 2015), the BH3-domain of Bim binds to the canonical groove of Bax. Our FRET data shows that BimL and Bax bind in solution and the binding is abolished by the 4E mutation in the Bim BH3-domain that eliminates the nonpolar interactions with the Bax groove. It is therefore expected that this Bim-Bax interaction is mediated by the BH3-domain and the groove, and according to the structure, a BMH molecule would be able to link a cysteine replacing Phe101 in the BH3-domain of Bim to a cysteine replacing Trp107 in the canonical groove of Bax. We thus synthesized the [35S]Met-labeled single-Cys Bim F101C and Bax W107C proteins in vitro, let them interact in solution, and subjected the sample to BMH crosslinking. When the products were analyzed by SDS-PAGE and phosphor-imaging two BMH-crosslinked products with molecular weights close to that of a BimL-Bax heterodimer were detected (Figure 9A, lane 4, indicated by open and closed triangles). The lower molecular weight band indicated by a closed triangle is the BMH-linked BimL-Bax heterodimer since it was absent in the control reactions when either the single-cysteine BimL or Bax was replaced by the respective cysteine-null (C0) protein (Figure 9A, lane 6 or 2). The higher molecular weight band indicated by an open triangle is the BMH-linked Bax homodimer since it was also present in the control reaction containing the single-cysteine Bax and the cysteine-null BimL (Figure 9A, lane 6). These results demonstrate the BMH crosslinking approach can detect the interaction of the BH3-domain of BimL with the canonical groove of Bax, and hence in principle it can be used to reveal the Bim CTS-binding site in Bax.

Sequence analysis predicts that similar to the BH3-domain the Bim CTS forms an amphipathic α-helix (Figure 6A). Sequence alignment revealed a high similarity between the CTS and the BH3-domain as both have the same hydrophobic residues at the h0, h1, h2 and h3 positions, and the same polar or charged residue at the h1+two or h2+one position (Figure 9D). The Bim BH3 residues at these positions make critical contacts with the Bax canonical groove that are important for Bax activation (Robin et al., 2015; Weber et al., 2007). To determine whether the Bim CTS binds to the same Bax groove as the Bim BH3-domain, we performed BMH crosslinking using a Bim W137C mutant that has a single cysteine near the C-terminus of the CTS. Like the Bim F101C mutant with the cysteine near the C-terminus of the BH3-domain, Bim W137C crosslinked to Bax W107C (Figure 9A, lane 10, indicated by a closed triangle), suggesting that the BH3-binding groove is also a binding site for the Bim CTS. Consistent with this interpretation, this BimL-Bax heterodimer specific crosslinking did not occur in the control reaction with either single-Cys protein substituted by the respective cysteine-null protein (Figure 9A, lane 8 or 12), unlike the Bax homodimer specific crosslinking that also occurred in the control reaction with the single-cysteine Bax and cysteine-null BimL (Figure 9A, lane 6). Reciprocal immunoprecipitation by BimL and Bax specific antibodies further identified the crosslinked BimL-Bax heterodimer from the cysteine in the Bim CTS or BH3-domain to the cysteine in the Bax groove (Figure 9—figure supplement 1).

To further define this noncanonical CTS-groove interaction, we repeated the crosslinking using other cysteine positions in the Bim CTS and additional cysteine mutants in the canonical groove in Bax. The only strong heterodimer specific crosslinking detected was between Bim M123C (a cysteine near the N-terminus of the CTS) and Bax W107C (Figure 9A, lane 14, indicated by a closed triangle). Since this Bax mutant was also crosslinked to the C-terminus of CTS via W137C, binding of the CTS to the groove seems to occur in either orientation. Additional weak but specific BimL to Bax crosslinking was detected from M123C to E69C and W137C to D98C providing further support for this flexible interaction (Figure 9—figure supplement 1).

To determine whether the physical interaction between the Bim CTS or BH3-domain and the Bax groove detected by the crosslinking is functional, we tested the effect of the L129E mutation in the Bim CTS or the 4E mutation in the Bim BH3-domain on the crosslinking because both mutations greatly inhibited the activation of Bax by BimL (Figure 6B). We found that the L129E mutation reduced the Bim CTS to Bax interaction with the binding groove detected by the Bim W137C or W123C to Bax W107C crosslinking (compare the closed triangle-indicated band in Figure 9B, lane 10 or 14 with that in Figure 9A, lane 10 or 14). Since we used Bim F101C to detect the BH3-domain interaction with the Bax groove, and the F101 was changed to E in the 4E mutant, we generated Bim F101C/3E mutant to assess the effect of the BH3 mutation. As expected, the 3E mutation abolished Bim F101C crosslinking to Bax W107C, and hence, the BH3-interaction with the groove (Figure 9C, the closed triangle indicates the band in lane two that disappeared in lane 4 when the 3E mutant was used). Surprisingly, mutation of the CTS also inhibited the BH3-interaction with the groove (Figure 9B, lane four vs. Figure 9A, lane 4), while the BH3 mutation also abolished the CTS interaction with the groove (Figure 9C, lane 12 vs. lane 10), suggesting that the two interactions are not independent. The BH3-domain may contribute more than the CTS to the overall protein-protein interaction based on the severity of the effect of mutations on the crosslinking and FRET (Figure 6B). Together, the crosslinking data from these loss-of-function Bim mutants demonstrate that the CTS and BH3-interactions with the groove detected between the soluble Bim and Bax proteins are functionally important for Bim mediated activation of Bax.

Bim CTS mutants that cannot activate Bax in vitro do not kill HEK293 cells

Together, our data suggests that specific residues within the Bim CTS are involved in different aspects of BimL function. Residue I125 is required for Bim to bind to mitochondria but is of lesser importance in activating Bax. In contrast, residues L129 and I132 are not required for BimL to bind Bax but are important for it to efficiently activate Bax. Finally, BimL-dCTS functions only to bind and inhibit Bcl-XL. The defined mechanism(s) of these mutants makes them useful for probing the differential sensitivity of HEK293 and MEF cells to expression of VBimL-dCTS as seen in Figure 1. Expression of the mutants in HEK293 cells by transient transfection revealed that similar to VBimL-dCTS, expression of either VBimL-L129E or VBimL-I132E was not sufficient to kill HEK293 cells, despite expression of either mutant being sufficient to kill the primed MEF cell line (Figure 10). In contrast, HEK293 cells were killed by expression of VBimL-I125E, albeit to a lesser extent than by VBimL (Figure 10). This result is consistent with our findings with purified proteins showing that the EC50 for liposome permeabilization by BimL-I125E was 100 nM compared to ~1 nM for BimL (Figure 6B). The activity of VBimL-I125E also demonstrates that BimL binding to membranes is not required to kill HEK293 cells as BimL-I125E does not bind membranes (Figure 6B). Together, this data suggests that unlike MEF cells, only mutants of BimL that can efficiently activate Bax kill HEK293 cells.

Discussion

The apoptotic activity of Bim in live cells is likely mediated by a combination of functions that result in both activation of Bax and inhibition of anti-apoptotic proteins. Unlike any of the known BH3-proteins or small molecule inhibitors, BimL-dCTS inhibits all of the major multi-domain Bcl-2 family anti-apoptotic proteins without activating Bax or Bak. Thus expression of this tool protein in cells enables new insight into the importance of the extent to which a cell depends on the expression of anti-apoptotic proteins for survival (Figure 1A, Figure 2B). Our results strongly suggest that the varying levels of apoptotic response of cell lines to BimL-dCTS reflect the extent to which that particular cell type is primed. Thus, HEK293 cells and mature neurons that are resistant to inhibition of Bcl-2, Bcl-XL and Mcl-1 but sensitive to activation of Bax, are functionally unprimed. Partial resistance to expression of BimL-dCTS suggests that the flow of Bcl-2 family proteins between different binding partners leads to differential levels of dependency on the activation of Bax to trigger apoptosis. To illustrate this, we have created a schema illustrating protein flow at the two extremes represented by fully unprimed and primed cells and the effects of mutations in the Bim CTS on regulating apoptosis (Figure 11). In the schema, flow is indicated by the different lengths of the equilibria arrows and illustrates the consequences of the various dissociation constants displayed in Figure 6B. While BimL efficiently recruits Bax to membranes and activates it, BimL-I125E does not bind to membranes and has reduced binding to Bax therefore this mutant activates Bax less efficiently than BimL (Figure 11A). BH3-proteins that do not efficiently activate Bax, such as BimL-L129E or BimL-I132E, do not result in MOMP in unprimed cells (Figure 11A) instead they interact primarily with anti-apoptotic proteins (illustrated here as Bcl-XL since it was possible to measure binding with this purified protein). The binding measurements in Figure 6B allow prediction of the outcome of more subtle differences in interactions for BimL and its mutants. For example, even though BimL-I125E activates Bax the concentration required is around 100 nM while the dissociation constant for Bcl-XL is less than 3 nM (Figure 6B) such that in cells BimL-I125E would preferentially bind and inhibit Bcl-XL rather than activate Bax (Figure 11B). While the CTS is necessary for Bim to activate Bax at physiologically relevant concentrations, membrane binding mediated by the CTS is not a prerequisite for interaction with Bax. Rather, binding to membranes increases subsequent Bax activation possibly through facilitating Bax conformational changes on the membrane (Figure 6B compare BimL, BimL-CTS2A and BimL-I125E). Thus, it is likely that in cells expressing endogenous Bim, binding to membranes contributes to the efficiency with which the protein kills cells. In addition, a recent publication suggests that membrane-bound Bim is more pro-apoptotic as it can dimerize using the LC1-motif by binding to DLC1 (Singh et al., 2017). Here, we observed that one reason that BimL-dCTS is less pro-apoptotic is loss of binding to membranes where it can activate Bax more efficiently. Future work will determine the relative importance of the CTS-mediated Bim binding to mitochondrial membranes and direct activation of Bax. In addition, it will be critical to determine the role for the DLC1-mediated Bim complex formation on binding anti-apoptotic proteins in the membranes. A key question is whether the resulting alterations of Bim interactions with other Bcl-2 family proteins that are mediated by BH3-domain, the CTS and LC1 region are regulated differently in different cell types. Nevertheless, restoring membrane binding to BimL-dCTS using a mitochondrial tail-anchor (BimL-dCTS-MAO) did not restore Bax activation, thus indicating that the Bim CTS does more than bind the protein to membranes . Additionally, our work suggests a newly characterized mechanistic distinction between Bim and tBid, as tBid requires membrane binding and a subsequent conformational change in order to bind and efficiently activate Bax (Lovell et al., 2008; Shamas-Din et al., 2013a) while BimL can activate Bax in solution via dual interactions with the Bim BH3 domain and CTS (Figures 3C, 8, 9 and 11).

The activities of the various Bim mutants analyzed here further suggest that specific residues in the Bim CTS enable physiological concentrations of Bim to activate Bax. That BimL-V124E, BimL-I125E and BimL-L129E all bind Bax in solution and in the presence of membranes with similar affinities yet vary functionally to trigger Bax-mediated liposome permeabilization by three orders of magnitude, suggests a specific role for this region in activation of Bax (Figure 6B) rather than the region simply increasing overall binding affinity. The situation is further complicated by another major role of the CTS of Bim in binding the protein to membranes. BimL-dCTS-MAO binds to mitochondria and liposomes yet is defective in activating Bax to permeabilize these membranes further suggesting a role for specific residues in the CTS binding to and activating Bax (Figure 7B). Such a role is consistent with our crosslinking data suggesting direct binding between the CTS of Bim and Bax (Figure 8) that is surprisingly mediated at least in part by the canonical BH3-binding groove in Bax (Figure 9). Furthermore, these CTS residues particularly L129 (which corresponds to L185 in BimEL) increased the affinity for Bim binding to Bcl-XL such that it conferred resistance to BH3 mimetic drugs (Liu et al., 2019). Nevertheless, it remains formally possible that changes in binding affinity coupled with alterations in effective off-rate due to membrane binding may also contribute to the activation of Bax by Bim. In addition to loss of interactions with the membrane BimL-dCTS still retains a greater propensity to activate Bax in comparison to Bim BH3-peptides (Compare Figure 3A with Sarosiek et al., 2013; Figure 4H). While 1 μM of BimL-dCTS was sufficient to activate Bax in our liposome release assay, over 10 μM of Bim BH3-peptide was required to achieve similar Bax activation. This result suggests that in addition to the BH3 domain and the CTS, other regions of Bim contribute to Bim’s pro-apoptotic function as previously suggested (Singh et al., 2017). Moreover, it is unclear to what extent other regions of Bim contribute to the BH3 region and the CTS adopting an optimal structure that can efficiently activate Bax. Similar to Bim, full length Bid cleaved by caspase is nearly 10-fold more active than a Bid-BH3 peptide in activating Bax, suggesting other regions outside the BH3-domain of Bid contribute to the interaction and/or activation of Bax. Thus, future studies of Bim and Bid should address what additional sequences other than the BH3-domains and C-terminal regions participate in Bax activation.

Currently, BH3-profiling is the technique used to assay the state of apoptotic priming for different tissue types, however, this technique requires the addition of BH3-peptides at high concentrations, and can only be performed on cells/tissues after permeabilization of the plasma membrane (Potter and Letai, 2016). As an alternative, we propose lentiviral delivery and expression of BimL-dCTS be performed on living cells (or tissue samples), with readouts currently being used to assay cell death such as Annexin V staining, condensed nuclei, PI staining of nuclei, etc. Recently, it was reported in adults that most tissues are unprimed (Sarosiek et al., 2017); however, the status of priming for different cell types that make up a single tissue may differ. In contrast, in tissue culture most cells are at least partially primed (Figure 1). We speculate that stress responses that result when fully or partially transformed cell lines are grown under non-physiological conditions (high glucose and oxygen, in the presence of serum and on plastic with abnormal stromal interactions) generally account for the dependence of these cell lines on continued expression of anti-apoptotic proteins. BH3-profiling can only provide an answer at the tissue level or for cell populations that can be isolated in sufficient quantities or easily cultured (Sarosiek et al., 2017). However, lentiviral delivery and expression of VBimL-dCTS in cells in co- or organoid-cultures, tissue slices and in vivo can provide a means to assay the level of dependence of individual cells on expression of anti-apoptotic Bcl-2 family proteins. This information could prove valuable to understanding which cell types may be most affected by small molecule BH3 mimetics as chemotherapeutics and for other drugs to better predict and prevent off-target toxicities that result in cell priming.

Overall, our data suggests a model in which the unusual CTS of Bim is not only required for binding to membranes but is directly involved in the activation of Bax. This interaction likely occurs via binding of the Bim CTS to the BH3-binding groove on Bax (Figure 9). However, the BH3-binding groove may not be the exclusive binding site for the Bim CTS. As seen in Figure 8C, the CTS of Bim photocrosslinks to 6H-Bax even in the presence of the L129E mutation. However, crosslinking is abolished though with the BH3 4E mutation. How Bim binding to the BH3 binding groove by both its BH3 region and CTS enables Bax activation is the subject of ongoing studies. Nevertheless, this function is crucial for BimL to kill unprimed cells. The CTS also increases the affinity of Bim for binding to Bcl-XL and Bcl-2 (Figure 11). The very much higher affinity of Bim for Bcl-XL and Bcl-2 compared to Bax ensures that in cells with excess anti-apoptotic proteins Bim is effectively sequestered and neutralized. In previous studies, we demonstrated that the additional affinity of the interaction of Bim with Bcl-XL and Bcl-2 provided by the Bim CTS is sufficient to dramatically reduce displacement of Bim by small molecule BH3 mimetics (Liu et al., 2019). Thus, regulation of apoptosis by Bcl-2 proteins is more complicated than presented in most current models. Moreover, the mutants and binding affinities described here provide the tools necessary for future studies of the relative importance of activation of Bax compared to inhibition of anti-apoptotic proteins in intact cells and in animals.

Materials and methods

Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Antibodyantibody to Cytochrome cIn house (Billen et al., 2008)(1:2000) Dilution
AntibodyDonkey anti-rabbit (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-150(1:10000) Dilution
AntibodyDonkey anti-mouse (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-152(1:10000) Dilution
AntibodyAntibody to BaxIn house (Zhu et al., 1996)Max6(1:1000) dilution
AntibodyAntibody to BimSanta Cruz BiotechnologyCat. #: sc-11425(1:50) dilution
Cell line (M. musculus)Baby Mouse Kidney(BMK)-DKO (Bax and Bak knockout) cellsOther (Degenhardt et al., 2002)Provided by Dr. Eileen White (Rutgers University)
Mycoplasma free, see Materials and methods
Cell line (H. sapiens)Cama-1RRID: CVCL_1115Provided by Dr. Linda Penn (University of Toronto). Mycoplasma Free, see Materials and methods
Cell line (H. sapiens)HEK293Other (Graham et al., 1977)RRID: CVCL_0045Provided by Dr. Frank Graham (McMaster University).
Mycoplasma Free
, see Materials and methods
Cell line (H. sapiens)HCT-116Other (Polyak et al., 1996)RRID: CVCL_0291Provided by Dr. Bert Vogelstein (John Hopkins University).
Mycoplasma Free
, see Materials and methods
Strain (M. musculus)Embryonic day 15 embryosThe Jackson LaboratoryC57BL/6JUsed for the preparation of primary cortical neurons and for purification of mitochondria, see Materials and methods.
Cell line (M. musculus)MEFOther (Pagliari et al., 2005)RRID: CVCL_U630Provided by Dr. Doug Green (St. Judes Children’s Research Hospital)
Mycoplasma Free, see Materials and methods
Chemical compound, drugDraq5ThermoFisher Scientific, Molecular probesCat. #62251Nuclear stain for live cell imaging
Chemical compound, drugHoescht 33258Cell signaling technologiesCat. # 4082SNuclear stain for live cell imaging
Chemical compound, drugTetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)ThermoFisher Scientific, Molecular probesCat. # T669Used to stain actively respiring mitochondria
Chemical compound, drugPropidium iodideBioshopCat. # PPO888.10Nuclear stain for dead cells
Chemical compound, drugAlexa 647-maleimideThermoFisher Scientific, Molecular probesCat. #: A20347Acceptor fluorophore in FRET experiments, when Alexa 568 is the donor.
Chemical compound, drugAlexa568-maleimideThermoFisher Scientific, Molecular probesCat. #. A20341Donor fluorophore in FRET experiments when Alexa 647 is the acceptor.
Chemical compound, drugANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt)ThermoFisher Scientific, Molecular probesA350Fluorophore used in liposome release assay (Billen et al., 2008)
Chemical compound, drugDPX (p-Xylene-Bis-Pyridinium Bromide)ThermoFisher Scientific, Molecular probesX1525Quencher used in liposome release assay (Billen et al., 2008)
Chemical compound, drugIANBD Amide (N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine)Molecular ProbesCat. #: D-2004Chemically reactive environment sensitive fluorophore. Reacts with Cysteine used to label proteins for in vitro study.
Chemical compound, drugPC (L-α-phosphatidylcholine)Avanti Polar LipidsCat. #: 840051CFor making liposomes, used 48% PC
Chemical compound, drugDOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine)Avanti Polar LipidsCat. #: 840035CFor making liposomes, used 10% DOPS
Chemical compound, drugPI (L-α-phosphatidylinositol)Avanti Polar LipidsCat. #: 840042CFor making liposomes, used 10% PI
Chemical compound, drugPE (L-α-phosphatidylethanolamine)Avanti Polar LipidsCat. #: 841118CFor making liposomes, used 28% PE
Chemical compound, drugTOCL, (18:1 Cardiolipin)Avanti Polar LipidsCat. #: 710335CFor making liposomes, used 4% TOCL
Chemical compound, drugbismaleimidohexane (BMH)Pierce22330Chemical crosslinker, Cysteine specific. Used for chemical crosslinking of Bim and Bax proteins, see Materials and methods.
Commercial assay or kitFugene HDPromegaCat. #: E2311Transfection reagent for mammalian cells
Commercial assay or kitTransIT-X2MirusCat. #: Mir 6003Transfection reagent for mammalian cells
Gene (H. sapiens)BaxIn house (Yethon et al., 2003)GI: L22473.1Expression plasmid for production of recombinant protein
Recombinant DNA reagent
(H. sapiens)
BaxIn house (Zhang et al., 2010; Zhang et al., 2016)GI: L22473.1For recombinant 6H-Bax protein used in photocrosslinking and for making Cys-null or single Cys recombinant protein in chemical crosslinking
Recombinant DNA reagent (H. Sapiens)Bcl-XLIn house (Ding et al., 2014)GI: Z23115.1For recombinant 6H-Bcl-XL protein used in photocrosslinking, membrane permeabilization, and protein-protein binding assays.
Gene (H. sapiens)BadIn house (Aranovich et al., 2012)GI: AB451254.1For expression of VBad in cells
Gene (M. musculus)BidIn house (Lovell et al., 2008)GI: NM_007544.4For recombinant cBid purification (
Gene (M. musculus)BimLThis paperGI: AAD26594.1This lab, plasmid # 2187, for recombinant BimL purificaton
Recombinant DNA reagent
(M. musculus)
BimLThis paperGI: AAD26594.1Dr. Lin lab, plasmidpSPUTK-BimL For the single-Cys proteins used in photo and chemical crosslinking
Recombinant DNA reagent
(M. musculus)
tBidIn house (Aranovich et al., 2012)GI: NM_007544.4For expression of VtBid in cells
OtherCell Carrier-384, Ultra platePerkinElmerCat. #: 6057300For mono-layer culturing and imaging cell lines
OtherGreiner Bio-one Cell culture microplate, 384 wellGreiner Bio-oneCat. 781090For culturing and imaging primary cortical neurons.
OtherNon-binding surface, 96-well plate, black with clear bottomCorningCat. #: 3881For recombinant protein and liposome assays. It is critical to use a non-binding plate.
OtherOpera PhenixPerkinElmerCat. #: HH14000000Automated confocal microscope. Used for imaging cell lines and primary cortical neurons.
OtherAnnexinV*Alexa647In House (Logue et al., 2009)Used for detecting phosphotidylserine externalization (Blankenberg et al., 1998)
OtherεANB-[14C]Lys-tRNALystRNA ProbesL-32Used for incorporation εANB-Lys into Bim protein using an in vitro translation system. The εANB-group is photoactive and generates a nitrene for photocrosslinking
Other[35S]MethioninePerkinElmerNEG009CUsed for incorporation [35S]Met into Bim and Bax proteins using an in vitro translation system for photo or chemical crosslinking,see Materials and methods.
Othertranscription/translation (TNT)-SP6 coupled wheat germ extract systemPromegaL4130Used for synthesis of Bim and Bax proteins for chemical crosslinking
Othermulti-purpose image scannerFuji FilmFLA-9000Used for phosphorimaging to detect radioactive proteins in gels
Software, algorithmGraphPad PrismSan Diego, CaliforniaVersion 6
RRID:SCR_002798
Scientific graphing program, used for curve fitting of in vitro data and to perform statistical analysis.
Software, algorithmImageJPMID: 17936939MBF - ImageJ for microscopy, Dr. Tony Collins (McMaster University)For band density measurements used to quantify Cytochrome c release from immunoblots.
Software, algorithmMulti GaugeFuji FilmVersion 3.0Used for processing and displaying phosphor-images
Recombinant DNA reagent (A. victoria)mVenus-pEGFP-C1OtherGI: KU341334.1Dr. Ray Truant (McMaster University). Backbone EGFP-C1 (Clonetech)

Protein purification

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Wild type and single cysteine mutants of Bax, Bcl-XL, and cBid were purified as described previously (Kale et al., 2014). cBid mutant 1 (cBidmt1) was purified with the same protocol used for cBid (Kale et al., 2014). Bad was purified as described previously (Lovell et al., 2008). His-tagged Bax and Bcl-XL proteins were purified as described previously (Ding et al., 2014)

His-tagged Noxa was expressed in E. coli strain BL21DE3 (Life Tech, Carlsbad, CA). E. coli cells were lysed by mechanical disruption with a French press. The cell lysate was diluted in lysis buffer (10 mM HEPES (7.2), 500 mM NaCl, 5 mM MgCl2, 0.5% CHAPS, 1 mM DTT, 5% glycerol, 20 mM Imidazole) and Noxa was purified by affinity chromatography on a Nickel-NTA column (Qiagen, Valencia, CA). Noxa was eluted with a buffer containing 10 mM HEPES (7.2), 300 mM NaCl, 0.3% CHAPS, 20% glycerol, 100 mM imidazole, dialyzed against 10 mM HEPES 7.2, 300 mM NaCl, 10% glycerol, flash-frozen and stored at −80°C.

Purification of BimL and single cysteine mutants of BimL was carried out as previously described (Liu et al., 2019). Briefly, cDNA encoding full-length wild-type murine BimL was introduced into pBluescript II KS(+) vector (Stratagene, Santa Clara, CA). Sequences encoding a polyhistidine tag followed by a TEV protease recognition site (MHHHHHHGGSGGTGGSENLYFQGT) were added to create an in frame fusion to the N-terminus of BimL. All the purified BimL proteins used here retained this tag at the amino-terminus. However, control experiments demonstrated equivalent activity of the proteins before and after cleavage with TEV protease (Data not shown). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis.

BimL was expressed in Arabinose Induced (AI) E. coli strain (Life Tech, Carlsbad, CA). E. coli were lysed by mechanical disruption with a French press. Proteins were purified from the cell lysate by affinity chromatography using a Nickel-NTA column (Qiagen, Valencia, CA), and eluted with a solution containing 20 mM HEPES pH7.2, 10 mM NaCl, 0.3% CHAPS, 300 mM imidazole, 20% Glycerol. The eluate was adjusted to 150 mM NaCl and applied to a High Performance Phenyl Sepharose (HPPS) column. Bim was eluted with a no salt buffer and dialyzed against 10 mM HEPES pH7.0, 20% glycerol, flash-frozen and stored at −80°C.

Protein labeling

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Single cysteine mutants of Bax, Bcl-XL, cBid and Bad were labeled with the indicated maleimide-linked fluorescent dyes as described previously (Pogmore et al., 2016; Kale et al., 2014; Lovell et al., 2008). Single cysteine mutants of Bim were labeled with the same protocol as cBid with the exception that the labeling buffer also contained 4M urea.

Bim binding to membranes

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Liposomes (100 nm diameter) with a lipid composition resembling MOM were prepared as described previously (Kale et al., 2014). Mouse liver mitochondria were isolated from Bak-/-mice as previously described (Pogmore et al., 2016). Liposomes and mitochondria were labeled with 0.5% and 2% mass ratios of DiD, respectively (Life Tech, Carlsbad, CA). The single-cysteine mutant of BimL, BimL Q41C, was labeled with Alexa568-maleimide and incubated with the indicated amount of unlabeled or DiD-labeled mitochondria or liposomes at 37°C for 1 hr. Intensities of Alexa568 fluorescence were measured in both samples as Funlabeled and Flabelled respectively using the Tecan infinite M1000 microplate reader. FRET, indicating protein-membrane interaction, was observed by the decrease of Alexa568 fluorescence when BimL bound to DiD labeled membranes compared to unlabeled membranes. FRET efficiency was calculated as described previously (Shamas-Din et al., 2013a). The data was fit to a binding model as described below. Lines of best fit were calculated using least squares in Graphpad Prism software.

Calculating the number of Bim molecules per Liposome

Step 1: Find the total number of Bim molecules in a 2 mL reaction

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Concentration of Bim: 5 nM in 2 mL reaction

Number of Bim (mole) = concentration (M) x volume (L)

  • = 5×10−9 M x 0.002 L

  • = 1×10−11 mol

Total number of Bim molecules in 2 mL reaction = Number of Bim (mole) x Avogadro’s Constant

  • = 1×10−11 mol x 6.02 × 1023 mol−1

  • = 6.02×1012 Bim molecules

Step 2: Find the total lipid surface area or the total number of liposome made from 1 mg lipid film

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The total number of lipid molecules in a liposome are given by the formula below

Ntotal = [4π(100 nm/2)2+4π((100 nm/2)–5) 2]/0.71nm2 = 80088.49 lipid molecules

Assuming that the average area of the lipid head group in our liposome is 0.71 nm2 (because it is made out of mostly PC).

Total lipid in 1 mg lipid film (in mole)=mass in gram/molecular wt (M.W) in gmol−1

  • = 0.001 g/ 804.95 gmol−1

  • = 1.24×10−6 mole of lipid

Total lipid molecules in 1 mg lipid film = 1.24×10−6 mol x 6.02 × 1023 mol−1

  • = 7.48 × 1017 lipid molecules

Molecular weight of our mitochondrial-like lipid film was calculated from the %Molar and molecular weight of individual lipid which was published in Kale et al. (2014) (Examining the Molecular Mechanism of Bcl-2 Family Proteins at Membranes by Fluorescence Spectroscopy).

Total number of liposomes = Total lipid molecule in 1 mg/Ntotal = 9.34 × 1012 liposomes.

Surface area of a liposome = 4πr2 = 4π(50 nm)2 = 10000π nm2

where r is the radius of our liposome.

Total lipid surface area = surface area of a liposome x total number of liposome = 2.93×1017 nm2

Step 3: Calculate the number of Bim per liposome or per surface area

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Number of Bim per liposome = Total number of Bim in a 2 mL reaction/Total number of liposome

  • = 6.02 × 1012 Bim molecules/9.34 × 1012 liposomes=0.64 Bim molecule per liposome

Number of Bim per surface area = Total number of Bim in a 2 mL reaction/Total lipid surface area

  • = 6.02 × 1012 Bim molecules/2.93 × 1017 nm2=2.05×10−5 Bim molecule per nm2

Membrane permeabilization

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Membrane permeabilization assays with liposomes encapsulating ANTS and DPX were performed as described previously (Kale et al., 2014). To measure permeabilization of BMK mitochondria, the indicated amounts of proteins were incubated with mitochondria (1 mg/mL) purified from BMK cells genetically deficient for Bax and Bak expressing mCherry fluorescent protein fused to the SMAC import peptide responsible for localization in the inter-membrane space. After incubation for 45 min at 37°C samples were centrifuged at 13,000 g for 10 min to separate the pellet and supernatant fractions and membrane permeabilization was calculated based on the mCherry fluorescence in each fraction (Shamas-Din et al., 2014). For mouse liver mitochondria, cytochrome c release was measured by immunoblotting as described previously (Pogmore et al., 2016; Sarosiek et al., 2013).

BH3 profiling

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Heavy membranes enriched in mitochondria were isolated as described previously (Pogmore et al., 2016; Brahmbhatt et al., 2016). Membrane fractions (1 mg/mL) were incubated with 500 nM of the specified BH3-proteins (Bim, Bad and/or Noxa). For E15 brain mitochondria, 0.5 mg/mL of membrane fractions were used and incubated with the indicated amounts of BH3-only proteins for 30 min at 37 °C. Membranes were pelleted by centrifugation at 13,000 g for 10 min and cytochrome c release was analyzed by immunoblotting using a sheep anti-cytochrome c antibody (Capralogics). Mitochondria from embryonic mouse brains for BH3profiling experiments were prepared from ~20 mouse embryos, E15 in age, following the same protocol used for liver mitochondria (Pogmore et al., 2016).

Protein-protein binding

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For FRET experiments, single cysteine mutants of cBid (126C), Bcl-XL (152C), Bax (126C), BimL (41C) and BimL mutants were purified and labeled with either Alexa 568-maleimide (donor) or Alexa 647-maleimide (acceptor) as specified. To determine binding a constant amount of donor protein was incubated with the indicated range of acceptor proteins and where specified liposomes or mitochondria. The intensity of Alexa568 fluorescence with unlabeled or Alexa647-labeled Bcl-XL was measured as Funlabeled or Flabeled, respectively, and FRET was calculated as described in Pogmore et al. (2016). All measurements were collected using the Tecan infinite M1000 microplate reader. Lines of best fit were calculated using least squares in Graphpad Prism software.

For each pair of proteins a dissociation constant (Kd) was measured in solution and with liposomes. Curves were fit to an advanced function taking into account change of the concentration of acceptor ([A]) when [A] is close to Kd:

[D] is the concentration of donor, F indicates the FRET efficiency with the concentration of acceptor as [A], Fmax is the maximum FRET efficiency in the curve (Pogmore et al., 2016).

Photo and chemical crosslinking of Bim to Bax or Bcl-XL

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To produce the proteins for crosslinking using in vitro systems, the DNA sequence encoding murine BimL Cys-null and Lys-null mutant without the His tag and TEV protease recognition site was excised from the pBluescript II KS(+) vector by restriction endonucleases NcoI and ClaI and inserted into the pSPUTK vector (Stratagene, Santa Clara, CA). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis to generate the single-Lys BimL or single-Cys mutants.

The photocrosslinking method for studying interactions among the Bcl-2 family proteins has been described in detail (Lin et al., 2019). Briefly, using the RNAs produced from the single-Lys Bim DNAs in the pSPUTK vector by an in vitro transcription system, [35S]Met-labeled BimL proteins with a single εANB-Lys incorporated at specific locations were synthesized in an in vitro translation system. 10 μL of the resulting BimL proteins were incubated at 37 °C for 1 hr with 1 μM of 6H-Bax or 6H-Bcl-XL protein and Bak-/-mouse liver mitochondria (0.5 mg/ml total protein and were resuspended in AT buffer with 80 mM KCl and supplemented with energy regenerating system as described previously Yamaguchi et al., 2007) in a 21 μL reaction adjusted by buffer A (110 mM KOAc, 1 mM Mg(OAc)2, 25 mM HEPES, pH 7.5). The mitochondrial and soluble fractions were separated by centrifugation at 13,000 g and 4 °C for 5 min, and the mitochondria were resuspended in 21 μL of buffer A. Both mitochondrial and soluble fractions were photolyzed to induce crosslinking via the ANB probe. The resulting samples were adjusted to 250 μL with buffer B (buffer A with 1% Triton X-100 and 10 mM imidazole) and incubated with 10 μL of Ni2+-chelating agarose at 4 °C for overnight. After washing the Ni2+-beads three times with 350 μL of buffer B and one time with 400 μL of PBS, the photoadducts of the radioactive BimL protein and the 6H-tagged Bax or Bcl-XL protein and other proteins bound to the Ni2+-beads were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphor-imaging.

For chemical crosslinking, [35S]Met-labeled single-Cys or Cys-null BimL and Bax proteins were synthesized from the respective mutant Bim and Bax DNAs in the pSPUTK vector using a transcription/translation (TNT)-coupled in vitro system (Promega, Madison, WI). The resulting BimL and Bax proteins, 2 μL each, were paired as indicated in Figure 9, and reduced by 11 μM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a 20 μL reaction adjusted with buffer A at 37 °C for 1 hr. The sample was diluted to 110 μL with buffer A and split evenly to two aliquots. For a ‘60 min’ BMH crosslinking reaction, one aliquot was incubated with 0.1 mM BMH and 6 mM EDTA at 25 °C for 60 min, and the reaction was stopped by incubation with 50 mM 2-mercaptoethanol at 25 °C for 15 min. For a ‘0 min’ control reaction, the other aliquot was incubated with 2-mecaptoethanol and EDTA for 15 min, and then with BMH for 60 min. The resulting samples were precipitated by trichloroacetic acid. The resulting protein pellets were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

To obtain the immunoprecipitation data in Figure 9—figure supplement 1, the indicated single-Cys BimL and Bax proteins produced by the TNT system, 4 μL each, were paired and reduced by TCEP. The sample was diluted to 260 μL with buffer A and crosslinked by BMH. The resulting sample was divided to two aliquots. The 85 μL or 170 μL aliquot was immunoprecipitated by Bax or Bim antibody, respectively. Thus, each aliquot was adjusted to 250 μL with IP buffer (100 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 1 mM PMSF, 1% (v/v) Triton X-100), and received Bax antibody (made in house, 1:1000 dilution) or Bim antibody (Santa Cruz Biotechnology, Dallas, TX, 1:50 dilution). The samples were rotated at 4 °C for overnight, and after receiving 25 μL of Protein G Sepharose (50% suspension in IP buffer), rotated for 2 more hours. After centrifugation at 2000 g for 0.5 min, the beads were washed three times with 400 μL of IP buffer and one time with 400 μL of 100 mM Tris pH 7.5 and 100 mM NaCl. The bound proteins were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

Measurement of cell death in response to expression of VBimL constructs

Request a detailed protocol

HEK293, BMK, MEF, and HCT116 cells were maintained at 37°C (5% v/v CO2) in dMEM complete [dMEM, 10% Fetal Bovine Serum, 1% essential amino acids (Gibco, Grand Island, NY)]. CAMA-1 were maintained the same environmental conditions but using dMEM/F12 (Gibco, Grand Island, NY). Cell lines were routinely confirmed to be mycoplasma-free using a PCR-based protocol as described by Hopert et al. (1993), and their authenticity was verified by short-tandem repeat (STR) profiling at The Centre for Applied Genomics (Toronto, ON, Canada) for human cells. Murine cell lines have not been authenticated. Cells were seeded in CellCarrier-Ultra 384-well plates (1000 cells/well for BMK and MEF, 2000 cells/well for HEK293 and HCT116, 3000 cells/well for CAMA-1). One day later, cells were transfected using FugeneHD (Promega, Madison, WI) with plasmids encoding Venus, or Venus-fused BimL constructs in an EGFP-C3 backbone. Cell culture medium was added to each reaction (50 µl/0.05 μg DNA) and the whole mix added to each well (50 µl/well) of a pre-aspirated 384-well plate of cells. After 24 hr, cells were stained with Draq5 and Rhodamine-labeled Annexin V and image acquisition was performed using the Opera QEHS confocal microscope (Perkin Elmer, Woodbridge, ON) with a 20x air objective. Untransfected cells and cells treated with 1 µg/mL staurosporine were used as negative and positive controls for Annexin V staining. Cells were identified automatically using software as described previously (Shamas-Din et al., 2013a). Intensity features were extracted using a script (dwalab.ca) written for Acapella high content imaging and analysis software (Perkin Elmer, Woodbridge, ON). Cells were scored as Venus or Annexin V positive if the Venus or Annexin V intensity was greater than the average intensity plus two standard deviations for the Venus or Annexin V channels in images of non-transfected cells. Cell death ascribed to the VBimL fusion proteins was quantified as the percentage of Venus-positive cells that were also Annexin V positive. For neuron cultures, cell segmentation using conventional methods could not be achieved due to complex cellular morphologies. Therefore, nuclei were first identified, then a ring region ~10% of nuclear area was drawn around each nucleus. Venus intensity was calculated for this ring region, representing the neuronal cell body, to determine if the neuron was expressing the Venus fluorescent protein.

Primary neuron cultures

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Primary cortical neuron cultures were prepared from embryonic day 15, C57BL/6J mouse embryos as previously described (Mergenthaler et al., 2012). All animal breeding and handling were performed in accordance with local regulations and after approval by the Animal Care Committee at Sunnybrook Research Institute, Toronto. Briefly, after separation from hippocampus and subcortical structures, cortices were washed twice with ice-cold PBS, digested with 1x trypsin for 15 min at 37°C, washed twice with ice-cold PBS and then resuspended with a flame-treated glass pipette in N-Medium (DMEM, 10% v/v FBS, 2 mM L-glutamine, 10 mM Hepes, 45 µM glucose). The dissociated cortices were gently pelleted by centrifugation (200 g for 5 min), N-media was removed, and neurons were resuspended and cultured in Neurobasal-Plus medium (ThermoFisher Scientific) supplemented with B27-Plus (ThermoFisher Scientific) and 1x Glutamax (ThermoFisher Scientific). Neurons were seeded at 5000 cells per well in a 384 well plate (Greiner µclear) after coating with poly-d-lysine (Cultrex). The medium was partially replaced on day 5 in culture with Neurobasal-Plus supplemented with B27-Plus and 1x Glutamax.

Lentivirus to express VBimL and other BimL mutants were cloned into the pTet-O-Ngn2-Puro construct with the Ngn2 gene cut out. This construct was a kind gift from Dr. Philipp Mergenthaler, Charité Universitätsmedizin Berlin. Primary neuron cultures were infected with both VBimL and rtTA lentiviral particles (~3 μL of each concentrated stock) on the day of seeding. 24 hr later, Neurobasal-Plus medium containing lentiviral particles was removed and replaced with fresh Neurobasal-Plus medium.

Doxycyline (ThermoFisher scientific) was added to 16 day in vitro old cultures of neurons at a concentration of 2 μg/mL to induce VBimL protein expression. 5 hr later, neurons were stained with 5 µM Draq5 (Thermofisher scientific) and 0.1 μM TMRE (Thermofisher scientific), then incubated for 30 min at 37 °C. Confocal microscopy was performed immediately after.

Lentiviral production

Request a detailed protocol

Each lentivirus was made using the following protocol adhering to biosafety level two procedures. On day 0, lentiviral vectors psPax2 (10 μg) and pMD2.G (1.25 μg) were mixed with 10 μg of desired VBimL lentiviral construct in 1000 μL of Opti-MEM media (ThermoFisher Scientific). Next, 42 μL of polyethylenimine (PEI) solution [1 mg/mL] was added, the mixture vortexed, then allowed to settle for 15 min at room temperature. After 15 min, 1.5 × 107 of resuspended HEK293 cells and the transfection solution were mixed and seeded onto a 100 mm culture dish with 10 mL of dMEM complete plus 10 μM of the caspase inhibitor Q-VD-Oph (Selleckchem), and left to incubate at 37 °C (5% v/v CO2) for 72 hr. On day 3, media containing lentiviral particles was filter sterilized using a 0.45 μm polyethersulfone filter, and mixed with polyethylene glycol (Bioshop) to achieve a final concentration of 10% (w/v). This was left to mix and precipitate the virus overnight at 4 °C. On day 4, the media was centrifuged for 1 hr at 1600 g, supernatant was then removed and the pellet was resuspended with 400 μL of Neurobasal-Plus media (no additives). Resuspended virus was then stored at −80 °C until needed.

Data availability

Data generated or analysed during this study are included in the manuscript and supporting files.

References

Источник: https://elifesciences.org/articles/44525

Ninja V

Capture your vision

The multi-award winning Atomos Ninja V Monitor-Recorder features a stunningly bright 5.2” 10-bit HDR display with 1000nits of output. The sleek aluminium body is crafted for maximum durability, weighing in at just 360g and measuring only 25mm thick – easily attaching to any camera rig.

Ninja V records directly from your camera’s sensor to a wide range of codecs: Apple ProRes, ProRes RAW, Avid DNx and H.265 (HEVC)*.This provides wide compatibility across camera types and edit software, giving you a new degree of creative control long before you press record.

* H.265 is an optional paid activation via my.atomos.com

The Ninja V takes all your creativity and brings it to life in the video you capture, regardless of whether you’re shooting cinematic productions, or vlogs for your social media channel. Whether you shoot HDR or regular SDR. Whether you use a professional cinema camera or a prosumer mirrorless camera.

Ninja V continues to innovate with regular free firmware updates, widely supported ProRes RAW recording, SDI connectivity with AtomX accessories and professional feature activations including H.265 recording.

Control what you see from Capture to SDR/HDR Delivery

    RECORDING

    Ninja V can record using the 10-bit H.265 (HEVC) codec by purchasing the upgrade at my.atomos.com for $99 (USD)

    H.265 adds even more versatility to the Ninja V, by adding a high-quality compressed codec option for recording. H.265 codec is used for the final delivery of high quality video projects with very small file sizes. HEVC was specifically created to provide support for UHD HDR deliverables with wide color gamuts, and enables you to easily share and allow play back of HDR content on a wide range of devices.

    If you’d like to know more about H.265 and Ninja V you can continue reading here, learn more about Ninja V, read more below or purchase now at my.atomos.com

    Read more

    MY.ATOMOS.COM

    The latest developments for Ninja V include

    • Multicam Switching

      with AtomX CAST

    • 5.9K ProRes RAW

      with selected cameras

    HDR / SDR

    State-of-the-art content creation

    Ninja V is the ultimate field monitor for the on-the-go film and video creator that values capturing to high quality industry standard codecs for SDR, HDR and next generation content delivery. The Ninja V’s state-of-the-art screen maintains clarity and color in the brightest of conditions and displays the widest dynamic range in HDR.

    The unique AtomHDR engine processes the incoming Log/RAW/HDR signals from your camera or device and displays in all its HDR glory on the Ninja V screen. There are matching profiles for a plethora of cameras, with more added as they emerge. Prefer to work in Rec.709? The Ninja V has you covered with the ability to load custom LUTs to match your workflow.

    UNLOCK PRO FEATURES


    RAW Recording
    from SDI Cameras

    Ninja V records ProRes RAW over SDI with compatible cameras using the AtomX SDI Module and paid upgrade of $99. The Activation Key for SDI RAW and HDMI RAW to SDI Video can be be purchased via your my.atomos.com account.

    These features bring even more versatility to the Ninja V, providing professional I/O options for a multitude of cameras – from entry level mirrorless cameras through to the latest compact cinema and high-end professional cameras. Read more about how Ninja V can expand your RAW eco-system.

    Ninja V Pro Kit

     

    MY.ATOMOS.COM
    <br/>RAW Recording<br>from SDI Cameras

    Instant Playback & Review

    Ninja V takes exactly what your camera sensor is capturing, enhances it and allows you to view and record it in amazing detail. Ninja V gives you the ability to record and instantly playback, review and tag your favourites takes to create playlists. This portable powerhouse gives you complete confidence in what you’re capturing, so you’re free to push creative boundaries more than ever before.

     

    Instant Playback & Review
    RECORD RAW

    Ninja V & ProRes RAW

    The number of cameras which now support RAW recording over HDMI on the Ninja V continues to grow. ProRes RAW combines the visual and workflow benefits of RAW video with the incredible real-time performance of ProRes. This format gives filmmakers enormous latitude when adjusting the look of their images, making it ideal for HDR workflows.

    COMPATIBLE CAMERAS
    Ninja V & ProRes RAW

    ProRes RAW Recording with Ninja V

    Learn more about Ninja V and the latest cameras at Atomos Academy

    Optional accessories for your Ninja V

    Essential accessories for capture, post-production and streaming

    Optional accessories for your Ninja V
    • AtomX SDI

      Input 12G/6G/3G/HD with the ability to toggle between input channel sources on SDI 1 / 2. Supporting up to 4kp60. Dual-link support for 1.5G/3G and 6G.

    • Battery Eliminator

      Power select Atomos monitor/recorder units

    • Sunhood

      Block excess reflections from washing out the displayed image

    • SSD

      Custom made for the new Atomos Ninja V monitor recorder

    • Docking Station

      Offload the drive contents direct to your computer

    • Connect

      Bridge recording and monitoring with streaming platforms such as YouTube, Twitch and OBS.

    • Monitor Mount

      Features a quick release plate, as well as 360° pan and 180° tilt functionality

    • DTap Cable

      D-Tap to DC Locked connector Barrel Coiled Cable for Battery Eliminator

    • AtomX Arm

      Attach your Atomos monitor to your camera cage or rig via a quick release workflow.

    PRO MONITORING

    Access All Areas

    The Ninja V offers a huge range of features that can enhance your workflow, from AtomOS Monitor Assist features like Focus Peaking, Zoom, VectorscopesFalse Color through to Safe Areas, Cine Guides and our AtomHDR processing engine that allows realtime Log>PQ/HLG previewing and more. Customizable settings & view modes — and instantly clear all overlays with a simple touch of the screen. Take control of your production at capture with Atomos Ninja V.

    Focus Peaking
    Customizable

    Weddings. Parties. Anything.

    Universally popular, Ninja V ticks more boxes for more filmmakers than any other 5” monitor on the market. And it will only get better. The Ninja V’s features continue to improve and evolve with every new AtomOS release. Not only will additional ProRes RAW capable cameras be supported, other new features are constantly being added for free.

    Recent additions include a wider range of frame guides (1:1, 4:5, 1:91:1 and 9:16) – perfect for social media content creation. A nine-grid overlay makes composing using the rule of thirds easy. The addition of an ARRI style false colour scale bolsters Ninja V’s impressive professional credentials.

    Weddings. Parties. Anything.

    Lock the shot with Ninja V’s monitoring and composition essentials.

    • Social Guides

      1:1 / 4:5 / 1:91:1 / 9:16

    • Safe Areas

      TV / Screen Guides

    • Frame Guides

      SMPTE standards

    Learn, Inspire, Create.

     

    Check out the latest content created using Atomos Ninja V.
    Features from Creators, tips from Pros and the latest innovations from Atomos.

    BROWSE ACADEMY
    SSD MEDIA

    Break through the recording time barrier

    Shooting 4K and above in advanced formats like ProRes, DNx and ProRes RAW requires storage media with ample volume, fast transfer times and high, sustained read and write speeds.

    AtomX SSDmini drives complete the Ninja V’s digital workflow. Smaller than conventional SATA SSDs they are an affordable professional alternative to recording to a camera’s internal memory cards. Offering up to 2TB storage, a sequential read speed of up to 550MB/s and write speed up to 500MB/s, these drives can record up to 150 minutes of 4K ProRes on a single drive. Measuring 8cm long, 7.5cm wide and weighing as little as 88g, the custom built drives neatly fit the Ninja V’s compact proportions.

    Break through the recording time barrier

    Fast. Reliable. Compact. Rugged.

      ProRes RAW

      Olympus E-M1X & E-M1 MIII with the Ninja V

      WATCH: Janne Amunét Director from Kauas Creative speaks first-hand of his experience shooting in ProRes RAW on the Olympus E-M1X & E-M1 MIII with the Ninja V. Understand the key features that make up the Olympus E-M1X & E-M1 MIII such as weatherproofing and image stabilisation and what this means when the mirrorless cameras are paired with the Ninja V to shoot in ProRes RAW.

       

      Related Posts

      FEATURE

      ProRes RAW on Z CAM + Ninja V

      WATCH: Go behind the scenes of UN/SEEN, an artistic abstract short film by filmmaker/director James Tonkin. UN/SEEN was shot on the Z CAM E2-F6 full-frame cinema camera and the Atomos Ninja V HDR monitor-recorder in Apple ProRes RAW.

      The behind the scenes video was shot on the Z CAM E2 and Ninja V in ProRes RAW. Watch the finished video below. Then view final project inHDR.

       

      ATOMOS ACADEMY

      ATOMX SYNC

      Making multi-camera mainstream

      You no longer need a huge budget or a highly skilled editor to produce perfectly synchronised, multicamera video. The optional AtomX SYNC module sits on the back of the Ninja V, integrating it into a Timecode Systems powered multi-camera sync network. The advanced long-range RF system is completely wireless, offering incredible accuracy and stability. Effortlessly synchronise your recordings with multiple camera and sound sources. With frame-accurate timecode embedded directly into each recording you can accurately edit together multiple video and audio sources effortlessly using automated functions in all popular editing software.

      LEARN MORE

      CALIBRATION

      Trust what you see

      Ninja V is your go anywhere monitoring system. Any camera, anywhere, any time of day, you can trust that you’re accurately seeing what you are recording. To ensure reliable and dependable accuracy, Atomos have partnered with calibration leader X-Rite to ensure all of our monitors operate seamlessly across Log/HDR capture, Post Production and HRD/SDR delivery. Ninja V is easy to calibrate, ensuring you are seeing your images accurately.

       

      X-RITE i1

      Unlock the full potential of your camera

      The diversity of codecs makes the Ninja V compatible with all major editing software packages. In terms of capture, virtually any HDMI or SDI source is supported, including cameras from Nikon, FujiFilm, Canon, Panasonic, Sony, Z-Cam, Olympus, RED and ARRI.

      Class leading monitoring technology

      • 60-240hz

        Recording frequency

      • Auto HDR

        Flags for TV setup

      Class leading monitoring technology
      • Zoom

      • Focus Peaking

      • LUTS

      • Zebra

      Gaming

      Record 4K HDR Gaming

      Ninja V is a stand-alone system for 4k UHD, HDR and high frame rate capture that eliminates expensive, complicated and unreliable PC setups. Ninja V enables simple recording, monitoring and instant review. Capture every detail in HDR and automatically include all the correct HDR flags ready for upload to YouTube. Ideal for games development testing, pre-release capture sessions or just to show off your skills!

      Ninja V

      Universally popular, Ninja V ticks more boxes for more filmmakers than any other 5” monitor on the market.

      $

      Please note:
      Specifications are subject to change without notice.
      All information correct at time of publishing.

      ° Paid activation. Available at myatomos.com

      FIND A RESELLER
      Ninja V

      Related products

      These Atomos product provide similar features. Try the COMPARE PRODUCTS feature to find out more.

      Spark a creative journey.
      Join the Atomos Community.

      Источник: https://www.atomos.com/products/ninja-v
      , , ¶

      Specifies an address to listen on for a stream (), datagram (), or sequential packet () socket, respectively. The address can be written in various formats:

      If the address starts with a slash (""), it is read as file system socket in the socket family.

      If the address starts with an at symbol (""), it is read as abstract namespace socket in the family. The "" is replaced with a character before binding. For details, see unix(7).

      If the address string is a single number, it is read as port number to listen on via IPv6. Depending on the value of (see below) this might result in the service being available via both IPv6 and IPv4 (default) or just via IPv6.

      If the address string is a string in the format "", it is interpreted as IPv4 address and port .

      If the address string is a string in the format "", it is interpreted as IPv6 address and port . An optional interface scope (interface name or number) may be specified after a "" symbol: "". Interface scopes are only useful with link-local addresses, because the kernel ignores them in other cases. Note that if an address is specified as IPv6, it might still make the service available via IPv4 too, depending on the setting (see below).

      If the address string is a string in the format "", it is read as CID on a port address in the family. The CID is a unique 32-bit integer identifier in analogous to an IP address. Specifying the CID is optional, and may be set to the empty string.

      Note that (i.e. ) is only available for sockets. (i.e. ) when used for IP sockets refers to TCP sockets, (i.e. ) to UDP.

      These options may be specified more than once, in which case incoming traffic on any of the sockets will trigger service activation, and all listed sockets will be passed to the service, regardless of whether there is incoming traffic on them or not. If the empty string is assigned to any of these options, the list of addresses to listen on is reset, all prior uses of any of these options will have no effect.

      It is also possible to have more than one socket unit for the same service when using , and the service will receive all the sockets configured in all the socket units. Sockets configured in one unit are passed in the order of configuration, but no ordering between socket units is specified.

      If an IP address is used here, it is often desirable to listen on it before the interface it is configured on is up and running, and even regardless of whether it will be up and running at any point. To deal with this, it is recommended to set the option described below.

      Specifies a file system FIFO (see fifo(7) for details) to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above.

      Specifies a special file in the file system to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above. Use this to open character device nodes as well as special files in and .

      Specifies a Netlink family to create a socket for to listen on. This expects a short string referring to the family name (such as or ) as argument, optionally suffixed by a whitespace followed by a multicast group integer. Behavior otherwise is very similar to the directive above.

      Specifies a POSIX message queue name to listen on (see mq_overview(7) for details). This expects a valid message queue name (i.e. beginning with ""). Behavior otherwise is very similar to the directive above. On Linux message queue descriptors are actually file descriptors and can be inherited between processes.

      Specifies a USB FunctionFS endpoints location to listen on, for implementation of USB gadget functions. This expects an absolute file system path of a FunctionFS mount point as the argument. Behavior otherwise is very similar to the directive above. Use this to open the FunctionFS endpoint . When using this option, the activated service has to have the and options set.

      Takes one of or . The socket will use the UDP-Lite () or SCTP () protocol, respectively.

      Takes one of , or . Controls the IPV6_V6ONLY socket option (see ipv6(7) for details). If , IPv6 sockets bound will be accessible via both IPv4 and IPv6. If , they will be accessible via IPv6 only. If (which is the default, surprise!), the system wide default setting is used, as controlled by , which in turn defaults to the equivalent of .

      Takes an unsigned integer argument. Specifies the number of connections to queue that have not been accepted yet. This setting matters only for stream and sequential packet sockets. See listen(2) for details. Defaults to SOMAXCONN (128).

      Specifies a network interface name to bind this socket to. If set, traffic will only be accepted from the specified network interfaces. This controls the socket option (see socket(7) for details). If this option is used, an implicit dependency from this socket unit on the network interface device unit is created (see systemd.device(5)). Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

      , ¶

      Takes a UNIX user/group name. When specified, all sockets and FIFO nodes in the file system are owned by the specified user and group. If unset (the default), the nodes are owned by the root user/group (if run in system context) or the invoking user/group (if run in user context). If only a user is specified but no group, then the group is derived from the user's default group.

      If listening on a file system socket or FIFO, this option specifies the file system access mode used when creating the file node. Takes an access mode in octal notation. Defaults to 0666.

      If listening on a file system socket or FIFO, the parent directories are automatically created if needed. This option specifies the file system access mode used when creating these directories. Takes an access mode in octal notation. Defaults to 0755.

      Takes a boolean argument. If yes, a service instance is spawned for each incoming connection and only the connection socket is passed to it. If no, all listening sockets themselves are passed to the started service unit, and only one service unit is spawned for all connections (also see above). This value is ignored for datagram sockets and FIFOs where a single service unit unconditionally handles all incoming traffic. Defaults to . For performance reasons, it is recommended to write new daemons only in a way that is suitable for . A daemon listening on an socket may, but does not need to, call close(2) on the received socket before exiting. However, it must not unlink the socket from a file system. It should not invoke shutdown(2) on sockets it got with , but it may do so for sockets it got with set. Setting is mostly useful to allow daemons designed for usage with inetd(8) to work unmodified with systemd socket activation.

      For IPv4 and IPv6 connections, the environment variable will contain the remote IP address, and will contain the remote port. This is the same as the format used by CGI. For , the port is the IP protocol.

      Takes a boolean argument. May only be used in conjunction with . If true, the specified special file is opened in read-write mode, if false, in read-only mode. Defaults to false.

      Takes a boolean argument. May only be used when . If yes, the socket's buffers are cleared after the triggered service exited. This causes any pending data to be flushed and any pending incoming connections to be rejected. If no, the socket's buffers won't be cleared, permitting the service to handle any pending connections after restart, which is the usually expected behaviour. Defaults to .

      The maximum number of connections to simultaneously run services instances for, when is set. If more concurrent connections are coming in, they will be refused until at least one existing connection is terminated. This setting has no effect on sockets configured with or datagram sockets. Defaults to 64.

      The maximum number of connections for a service per source IP address. This is very similar to the directive above. Disabled by default.

      Takes a boolean argument. If true, the TCP/IP stack will send a keep alive message after 2h (depending on the configuration of ) for all TCP streams accepted on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults to .

      Takes time (in seconds) as argument. The connection needs to remain idle before TCP starts sending keepalive probes. This controls the TCP_KEEPIDLE socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 7200 seconds (2 hours).

      Takes time (in seconds) as argument between individual keepalive probes, if the socket option has been set on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 75 seconds.

      Takes an integer as argument. It is the number of unacknowledged probes to send before considering the connection dead and notifying the application layer. This controls the TCP_KEEPCNT socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 9.

      Takes a boolean argument. TCP Nagle's algorithm works by combining a number of small outgoing messages, and sending them all at once. This controls the TCP_NODELAY socket option (see tcp(7)). Defaults to .

      Takes an integer argument controlling the priority for all traffic sent from this socket. This controls the socket option (see socket(7) for details.).

      Takes time (in seconds) as argument. If set, the listening process will be awakened only when data arrives on the socket, and not immediately when connection is established. When this option is set, the socket option will be used (see tcp(7)), and the kernel will ignore initial ACK packets without any data. The argument specifies the approximate amount of time the kernel should wait for incoming data before falling back to the normal behavior of honoring empty ACK packets. This option is beneficial for protocols where the client sends the data first (e.g. HTTP, in contrast to SMTP), because the server process will not be woken up unnecessarily before it can take any action.

      If the client also uses the option, the latency of the initial connection may be reduced, because the kernel will send data in the final packet establishing the connection (the third packet in the "three-way handshake").

      Disabled by default.

      , ¶

      Takes an integer argument controlling the receive or send buffer sizes of this socket, respectively. This controls the and socket options (see socket(7) for details.). The usual suffixes K, M, G are supported and are understood to the base of 1024.

      Takes an integer argument controlling the IP Type-Of-Service field for packets generated from this socket. This controls the socket option (see ip(7) for details.). Either a numeric string or one of , , or may be specified.

      Takes an integer argument controlling the IPv4 Time-To-Live/IPv6 Hop-Count field for packets generated from this socket. This sets the / socket options (see ip(7) and ipv6(7) for details.)

      Takes an integer value. Controls the firewall mark of packets generated by this socket. This can be used in the firewall logic to filter packets from this socket. This sets the socket option. See iptables(8) for details.

      Takes a boolean value. If true, allows multiple bind(2)s to this TCP or UDP port. This controls the socket option. See socket(7) for details.

      , , ¶

      Takes a string value. Controls the extended attributes "", "" and "", respectively, i.e. the security label of the FIFO, or the security label for the incoming or outgoing connections of the socket, respectively. See Smack.txt for details.

      Takes a boolean argument. When true, systemd will attempt to figure out the SELinux label used for the instantiated service from the information handed by the peer over the network. Note that only the security level is used from the information provided by the peer. Other parts of the resulting SELinux context originate from either the target binary that is effectively triggered by socket unit or from the value of the option. This configuration option applies only when activated service is passed in single socket file descriptor, i.e. service instances that have standard input connected to a socket or services triggered by exactly one socket unit. Also note that this option is useful only when MLS/MCS SELinux policy is deployed. Defaults to "".

      Takes a size in bytes. Controls the pipe buffer size of FIFOs configured in this socket unit. See fcntl(2) for details. The usual suffixes K, M, G are supported and are understood to the base of 1024.

      , ¶

      These two settings take integer values and control the mq_maxmsg field or the mq_msgsize field, respectively, when creating the message queue. Note that either none or both of these variables need to be set. See mq_setattr(3) for details.

      Takes a boolean value. Controls whether the socket can be bound to non-local IP addresses. This is useful to configure sockets listening on specific IP addresses before those IP addresses are successfully configured on a network interface. This sets the / socket option. For robustness reasons it is recommended to use this option whenever you bind a socket to a specific IP address. Defaults to .

      Takes a boolean value. Controls the / socket option. Defaults to .

      Takes a boolean value. This controls the socket option, which allows broadcast datagrams to be sent from this socket. Defaults to .

      Takes a boolean value. This controls the socket option, which allows sockets to receive the credentials of the sending process in an ancillary message. Defaults to .

      Takes a boolean value. This controls the socket option, which allows sockets to receive the security context of the sending process in an ancillary message. Defaults to .

      Takes a boolean value. This controls the , , or socket options, which enable reception of additional per-packet metadata as ancillary message, on , , and sockets. Defaults to .

      Takes one of "", "" (alias: "", "") or "" (alias: ""). This controls the or socket options, and enables whether ingress network traffic shall carry timestamping metadata. Defaults to .

      Takes a string value. Controls the TCP congestion algorithm used by this socket. Should be one of "", "", "", "" or any other available algorithm supported by the IP stack. This setting applies only to stream sockets.

      , ¶

      Takes one or more command lines, which are executed before or after the listening sockets/FIFOs are created and bound, respectively. The first token of the command line must be an absolute filename, then followed by arguments for the process. Multiple command lines may be specified following the same scheme as used for of service unit files.

      , ¶

      Additional commands that are executed before or after the listening sockets/FIFOs are closed and removed, respectively. Multiple command lines may be specified following the same scheme as used for of service unit files.

      Configures the time to wait for the commands specified in , , and to finish. If a command does not exit within the configured time, the socket will be considered failed and be shut down again. All commands still running will be terminated forcibly via , and after another delay of this time with . (See in systemd.kill(5).) Takes a unit-less value in seconds, or a time span value such as "5min 20s". Pass "" to disable the timeout logic. Defaults to from the manager configuration file (see systemd-system.conf(5)).

      Specifies the service unit name to activate on incoming traffic. This setting is only allowed for sockets with . It defaults to the service that bears the same name as the socket (with the suffix replaced). In most cases, it should not be necessary to use this option. Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

      Takes a boolean argument. If enabled, any file nodes created by this socket unit are removed when it is stopped. This applies to sockets in the file system, POSIX message queues, FIFOs, as well as any symlinks to them configured with . Normally, it should not be necessary to use this option, and is not recommended as services might continue to run after the socket unit has been terminated and it should still be possible to communicate with them via their file system node. Defaults to off.

      Takes a list of file system paths. The specified paths will be created as symlinks to the socket path or FIFO path of this socket unit. If this setting is used, only one socket in the file system or one FIFO may be configured for the socket unit. Use this option to manage one or more symlinked alias names for a socket, binding their lifecycle together. Note that if creation of a symlink fails this is not considered fatal for the socket unit, and the socket unit may still start. If an empty string is assigned, the list of paths is reset. Defaults to an empty list.

      Assigns a name to all file descriptors this socket unit encapsulates. This is useful to help activated services identify specific file descriptors, if multiple fds are passed. Services may use the sd_listen_fds_with_names(3) call to acquire the names configured for the received file descriptors. Names may contain any ASCII character, but must exclude control characters and "", and must be at most 255 characters in length. If this setting is not used, the file descriptor name defaults to the name of the socket unit, including its suffix.

      , ¶

      Configures a limit on how often this socket unit my be activated within a specific time interval. The may be used to configure the length of the time interval in the usual time units "", "", "", "", "", … and defaults to 2s (See systemd.time(7) for details on the various time units understood). The setting takes a positive integer value and specifies the number of permitted activations per time interval, and defaults to 200 for sockets (thus by default permitting 200 activations per 2s), and 20 otherwise (20 activations per 2s). Set either to 0 to disable any form of trigger rate limiting. If the limit is hit, the socket unit is placed into a failure mode, and will not be connectible anymore until restarted. Note that this limit is enforced before the service activation is enqueued.

      Источник: https://www.freedesktop.org/software/systemd/man/systemd.socket.html

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      The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

      Abstract

      When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

      Introduction

      Most intracellular functions are carried out by proteins, assembled as chains of peptide-bond linked amino acid (aa) residues on large ribonucleoprotein particles called ribosomes. The aa-sequences are specified by information stored as deoxyribonucleic acid (DNA) sequences in the genome and transcribed into sequences of messenger RNAs (mRNAs). The mRNAs are translated into aa-sequences with the help of transfer RNAs (tRNAs) reading any of their 61 aa-encoding ribonucleotide triplets (codons). In termination of translation, the complete protein is released from the ribosome by a class-1 release factor (RF) recognizing one of the universal stop codons (UAA, UAG, and UGA), signaling the end of the amino acid encoding open reading frame (ORF) of the mRNA. There are two RFs in bacteria, RF1 and RF2, one in eukarya, eRF1. RF1 and RF2 read UAA, UAG, and UAA, UGA, respectively, while the omnipotent eRF1 reads all three stop codons. Each stop codon in the decoding center (DC) is recognized by a stop-codon recognition (SCR) motif in a class-1 RF, and all RFs have a peptidyl transfer center (PTC)-binding GGQ motif, named after its universal Gly–Gly–Gln triplet (GGQ), for coordinated ester bond hydrolysis in the P-site bound peptidyl-tRNA. The crystal structures of free RF1 and RF2 have a distance between the SCR and GGQ motifs of about 20 Å1,2, much shorter than the 70 Å separating DC and PTC in the bacterial 70S ribosome. This distance discrepancy made the expected coordination between SCR and ester bond hydrolysis enigmatic. The crystal structure of free eRF1 has, in contrast, about 70 Å between its SCR and GGQ motifs, a distance close to the 80 Å between the DC and PTC of the 80S ribosome in eukarya3. Further cryo-EM work showed that ribosome-bound RF1 and RF2 have extended structures4,5, facilitating coordinated codon recognition in DC and ester bond hydrolysis in PTC. Subsequent high-resolution X-ray crystal6,7,8,9,10,11,12,13 and cryo-EM14,15,16,17,18,19 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF220, eRF121, and GGQ-mediated ester bond hydrolysis22.

      If the compact forms of free RFs in the crystal1,2 are physiologically relevant, it would mean that eubacterial RFs are in the compact form upon A-site entry (pre-accommodation state) and assume the extended form (accommodation state) in a stop-codon dependent manner. The relevance is indicated by a compact crystal structure of RF1 in a functional complex with its GGQ-modifying methyltransferase23,24, although SAXS data indicated free RF1 to be extended in bulk solution25. At the same time, SAXS data from T. thermophilus RF2 free in solution suggested a compact form for the factor or, possibly a mixture of compact and extended forms26. The existence of a RF-switch from a compact, free form to an extended ribosome-bound from would make high-resolution structures of these RF-forms necessary for a correct description of the stop-codon recognition process, hitherto based on post-termination ribosomal complexes27,28.

      Indirect evidence for rapid conformational activation of RF1 and RF2 after A-site binding has been provided by quench-flow based kinetics22, and in a series of recent FRET experiments Joseph and collaborators showed free RF1 to be in a compact form29, compatible with the crystal forms of RF11 and RF22, but in an extended form when bound to the A site of the stop-codon programmed ribosome29. Ribosome-bound class-1 RFs in the compact form has been observed together with alternative ribosome-rescue factor A (ArfA) in ribosomal rescue complexes, which lack any codon in the A site14,15. Very recently, Svidritskiy and Korostelev6 used X-ray crystallography in conjunction with the peptidyl transfer-inhibiting antibiotic blasticidin S (BlaS) to capture a mutated, hyper-accurate variant of RF1 in the stop codon-programmed termination complex. They found RF1 in a compact form, which they used to discuss stop-codon recognition in conjunction with large conformational changes of the RFs. It seems, however, that this BlaS-halted ribosomal complex is in a post-recognition state (i.e., stop-codon recognition motif has the same conformation as in the post-accommodation state in DC) but before RF-accommodation in the A site, making its relevance for on-pathway stop-codon recognition unclear (However, see also below!).

      Here, in contrast, we use time-resolved cryo-EM30,31,32,33,34 for real-time monitoring of how RF1 and RF2 ensembles change from compact to extended RF conformation in the first 100 ms after RF-binding to the pre-termination ribosome. These compact RF-structures, originating from short-lived ribosomal complexes previously out of reach for structural analysis, are seen at near-atomic resolution (3.5–4 Å). The time-dependent ensemble changes agree qualitatively with accompanying and previous22 quench-flow studies. We discuss the role of the compact structures of RF1 and RF2 for fast and accurate stop-codon recognition in translation termination.

      Results

      Kinetics study predicts compact RF1/RF2 exist at 20 ms

      We assembled a UAA-programmed release complex, RC0, with tripeptidyl-tRNA in the P site, and visualized its structure with cryo-EM (Methods and Supplementary Fig. 1). The RC0 displays no intersubunit rotation, and the tripeptide of its P-site tRNA is seen near the end of the peptide exit tunnel (Supplementary Fig. 1). The mRNA of the DC is disordered, but the overall resolution of the RC0 is high (2.9 Å). Apart from a small fraction of isolated ribosomal 50S subunits, the RC0 ensemble is homogeneous (Supplementary Fig. 1). We used quench-flow techniques to monitor the time evolution of the class-1 RF-dependent release of tripeptide from the peptidyl-tRNA with UAA-codon in the A site after rapid mixing of RC0 with RF1 or RF2 at rate-saturating concentrations (kcat-range)22 (Fig. 1a). The experiments were performed at pH values from 6 to 8 units, corresponding to [OH] variation in the 0.25–2.5 µM range (Supplementary Figs 2 and  3). The results are consistent with the existence of a two-step mechanism, in which a pH-independent conformational change (rate constant kconf) is followed by pH-dependent ester bond hydrolysis (see Methods). We estimate kconf as 18 ± 3 s−1 for RF1 and 11 ± 1 s−1 for RF2 at 25 °C, which approximates the effective incubation temperature for the time-resolved cryo-EM experiments (Supplementary Figs 2 and  3).

      Time evolution of ribosome ensembles in termination of translation a. Cartoon visualization of the pathway from free release complex to peptide release. Compact class 1 release factor (RF) binds to RF-free ribosomal release complex (RC0) and forms the RC·RFcompact complex with compounded rate constant ka·[RFfree]. Stop codon recognition induces conformational change in the RF which brings the ribosome from the RC·RFcompact to the RC·RFextended complex with rate constant kconf. The ester bond between the peptide and the P-site tRNA is hydrolyzed with rate constant khydr. b Predicted dynamics of peptide release with conformational change in RF1. We solved the form z download - Free Activators differential equations associated with termination according to the scheme in a with association rate constant ka = 45 µM−1s−1, [RF1free] = 3 µM, kconf = 18 s−1 and khydr = 2 s−1 (Supplementary Fig. 2) and plotted the fractions of ribosomes in RC0, RC·RFcompact and RC·RFextended forms (y-axis) as functions of time (x-axis). Green dot lines, RC0; red solid lines, RC·RFcompact; blue dash lines, RC·RFextended. c Predicted dynamics of peptide release with conformational change in RF2. The fractions of ribosomes in different release complexes were obtained in the same way as b with the rate constants ka = 17 µM−1 s−1, [RF2free] = 3 µM, kconf = 11 s−1 and khydr = 2.7 s−1 (Supplementary Fig. 3). d, e The populations of release complexes containing compact conformation and extended conformation of RF1 (d) and RF2 (e) at the 24 ms, 60 ms and endnote 9.1 product key - Free Activators incubation time points as obtained by time-resolved cryo-EM after 3D classification of the particle images

      Full size image

      From the quench-flow data, we predicted that the ensemble fraction of the compact RF1/2 form would be predominant at 24 ms, and much smaller at 60 ms (Fig. 1b, c). These predictions are in qualitative agreement with the time-resolved cryo-EM data, which show a somewhat faster conformational transition than in the quench-flow experiments (Fig. 1d, e). The difference in termination rates is, we suggest, due to a local temperature increase by friction inside the microfluidic chip. We first focus on the cryo-EM structures of RF1, and then highlight the few structural differences between RF1 and RF2.

      Time-resolved cryo-EM analysis

      At 24 ms reaction time, 25% of ribosome-bound RF1 is in the compact form in what we name the pre-accommodation state of the ribosome (Fig. 1d). The 70S part of the complex is similar to that of the pre-termination complex preceding RF-binding, but there is an additional A-site density belonging to RF1 (Fig. 2a). In pre-accommodation state of the ribosome, domain III of RF1 is 60–70 Å away from the PTC, in a similar relative orientation as in the crystal forms of the free factors1,2 (Supplementary Fig. 4) and significantly differing from that in the post-accommodated state of the terminating ribosome5. The loop that contains the GGQ motif of RF1 is positioned at the side of the β-sheet of domain II (near aa 165–168), facing the anticodon-stem loop and the D stem of the P-site tRNA (Fig. 2a, c).

      Cryo-EM structures of E.coli 70S ribosome bound with release factor 1. a Pre-accommodated ribosome complex bound with RF1 in a compact conformation. b Accommodated ribosome complex bound with RF1 in an extended conformation. Light blue: 50S large subunit; light gold: 30S small subunit; green: tripeptide; orange: P-tRNA; pink: mRNA; red: compact RF1; and dark blue: extended RF1. c, d Positions of domain III of ribosome-bound RF1 in pre-accommodated ribosome complex (c) and accommodated RF1-ribosome complex (d) relative to mRNA (pink), P-tRNA (orange) and tripeptide (green). e, f Close-up views of the upper peptide exit tunnel, showing tripeptide (green) in pre-accommodated ribosome complex (e) and accommodated ribosome complex (f)

      Full size image

      At 60 ms reaction time the RF1-bound ribosome ensemble is dominated by the extended form of RF1 (Fig. 2b, d). We term the ribosome complex with extended RF1 the accommodated RF1-ribosome complex. It contains density for the tripeptide in the exit tunnel, indicating that at 60 ms the peptide has not been released from the ribosome (Fig. 2e, f and Supplementary Fig. 5). At a much later time-point (45 s) the tripeptide density is no longer present in the exit tunnel of the accommodated RF-ribosome complex. Precise estimation of the time evolution of tripeptide dissociation from the ribosome would require additional time points. Of particular functional relevance would be estimates of the dc unlocker cracked apk of dissociation of longer peptide chains from the exit tunnel.

      The most striking difference between the compact and extended conformation of ribosome-bound RF1 is the position of the GGQ of domain III. As RF1 switches its conformation from the compact to the extended form, the repositioning of domain III places the catalytic GGQ motif within the PTC, and adjacent to the CCA end of the P-site tRNA (Fig. 2c, d). The extended form of RF1 has a similar conformation as found in the previous studies7,10,12,13,35,36.

      Similar to the case of sense-codon recognition by tRNA, three universally conserved DC residues, A1492, A1493, and G530 of the ribosome’s 16S rRNA undergo key structural rearrangements during translation termination. In the RF-lacking termination complex, A1492 of helix 44 in 16S rRNA stacks with A1913 of H69. A1493 is flipped out and stabilizes the first two bases in the A-site stop codon. G530 stacks with the third base A in the stop codon. In the presence of RF, whether compact or extended, A1492 is flipped out towards G530 and interacts with the first two stop-codon bases. A1493 stacks with A1913, which is in close contact with A1492 in the RF-lacking termination complex. G530 stacks with the third stop-codon base (Fig. 3a, b).

      Interaction of RF1 with the ribosomal decoding center. a, b Structures of the ribosomal decoding center in pre-accommodated ribosome complex (a) and accommodated ribosome complex (b). Red: compact RF1; dark blue: extended RF1. c, d Conformations of switch loop in pre-accommodated ribosome complex (c) and accommodated ribosome complex (d). Gold: A1492 and A1493; and orange: S12

      Full size image

      The switch loop, which was previously proposed to be involved in inducing a conformational change of RF110,36, shows no interaction with protein S12 or 16S rRNA in the compact form of RF1 (Fig. 3c) whereas in the extended form of RF1, the rearranged conformation of the switch loop is stabilized by interactions within a pocket formed by protein S12, the loop of 16S rRNA, the β-sheet of domain II, and A1493 and A1913 (Fig. 3b, d). Shortening the switch loop (302–304) resulted in a substantially slower, rate-limiting step in peptide release37, which indicates that the switch loop plays a role in triggering the conformational change of RF1.

      Similar experiments were carried out for RF2 at 24 ms, 60 ms and 5 h reaction times. RF2 undergoes a conformational change from compact to expanded form similar to that of RF1 (Fig. 1e). As in the case of RF1, the switch loop of RF2 makes no contact with protein S12 or 16S rRNA. In the extended form of RF2, A1492 is flipped out from helix 44 (h44) of 16S rRNA and stacks on the conserved Trp319 of the switch loop, stabilizing the extended conformation of RF2 on the ribosome.

      The ribosome complexes with RF1/2 bound in compact conformation seen here are distinct from those reported for the ribosome rescue complex, in which ArfA is bound in the A site lacking a stop codon14,15. In our structures, the conformation of the conserved 16S rRNA residues in the DC (A1492, A1493, and G530) is similar to the classical termination configuration10. In contrast, in the presence of ArfA, these residues adopt conformations known from sense-codon recognition14,15. It suggests that the compact RFs bind to the A site regardless of the conformation of the DC. The conformational change of RFs is likely due to the changes in the switch loop triggered by its interaction with protein S12 and 16S rRNA. This interaction is disrupted by the mutation A18T of ArfA, hence leaving RFs in the compact conformation15.

      Our ribosome complexes with RF1/2 are also distinct from a recent ribosome complex with compact RF1, reported by Svidritskiy and Korostelev6. Shortening of the switch loop, combined with the addition of the antibiotic BlaS which prevents the GGQ motif from reaching the PTC, stabilizes ribosome-bound RF1 in a compact conformation6, distinct from the transient, compact RF1-structure observed here. In our structure, the SCR between the β4–β5 strands on domain II are bound loosely to the A site (Fig. 3a). In the BlaS-halted compact RF1 structure6, in contrast, the stop codon-recognition motif of RF1 has moved further into the A site by 5 Å, to a position almost identical to that of the fully accommodated, extended structure of RF1. The functional role of their structure is not immediately obvious, but if it can be interpreted as an authentic transition state analogue, the roles of our respective complexes would be complementary. We previously found that high accuracy of stop signal recognition depends on smaller dissociation constant (Km-effect) and larger catalytic rate constant (kcat-effect) for class-1 RF reading of cognate stop codons compared to near-cognate sense codons38. The Km-effect contributes by factors from 100 to 3000 and the kcat-effect by factors from 2 to 3000 to the overall termination accuracy values in the 103–106 range38. Accordingly, the present structure may represent binding of RFs in a transient state where rapid and codon-selective dissociation rates are responsible for the accuracy factor due to the Km-effect. Furthermore, Korostelev's structure6, with its comparatively deep interaction between the cognate stop codon and SCR center, could mimic the authentic transition state on the path from compact to the extended form of the RF. Accordingly, Korostelev's structure may illustrate additional selectivity due to the kcat-effect. To test these hypotheses, molecular computations28 based on our respective RF structures could be used to compare their stop codon selectivities with those of RFs in the post-termination state of the ribosome20.

      In a recent paper on the role of RF3 in the dissociation of the release factors RF1 and RF239, the authors observed an interaction between domain I of RF1 and L7/L12 proteins, which assists the binding of RF1, as supported by complementary functional analysis using L7/L12 deletion mutants. However, such an interaction is not observed in our structures. Another recently published study using smFRET40 reported two states of the termination complex, non-rotated and rotated, in apparent contradiction to our results as we only found one, non-rotated state. The rotated-state subpopulation observed by Adio et al.40 may represent the post-termination ribosome unbound to RF1/RF2, as also suggested by previous single-molecule work from Puglisi and Gonzalez labs41,42.

      Discussion

      During translation termination, the release of the nascent peptide must be strictly coordinated with the recognition of a stop codon at the A site. Our cryo-EM analysis shows that in the presence of a class-1 RF the bacterial ribosome adopts several states. After rapid addition of RF1 or RF2 to a ribosomal termination complex with tripeptidyl-tRNA attached at the P site, we first observe the pre-accommodated RF-ribosome complex (compact form of RF) at 24 ms with the peptide attached to the P-site tRNA. This, we suggest, is the first step in the termination reaction. Second, at 60 ms, we observe the accommodated RF-ribosome complex with the extended form of RF and the tripeptide in the exit tunnel. Third, at a much later time point, we observe the post-accommodated RF-ribosome complex, with the extended form of RF without tripeptide in the exit tunnel (Supplementary Fig. 5). These pieces of evidence from our time-resolved experiments clearly reflect the sequence of events in termination of bacterial protein synthesis. A structure-based model for the stepwise interaction between ribosome and RF and the release of the nascent peptide from the termination complex during the translation termination process is presented in Fig. 4. It shows how the ribosome traverses (1) the pre-termination state with the stop codon at the A site, (2) the initial binding state (RF compact; pre-accommodated RF-ribosome complex), (3) the open catalytic state (RF open/extended; accommodated RF-ribosome complex) and (4) the state after peptide release. We suggest that the selective advantage of the compact RF-form is that it allows for rapid factor binding into and dissociation from an accuracy maximizing pre-accommodation state.

      Structure-based model. The sequence of states is (1) the termination complex with the stop codon at the A site, (2) the initial binding state (RF compact; “pre-accommodated RF-ribosome complex”), (3) the open catalytic state (RF open/extended; “accommodated RF-ribosome complex”) and (4) the state after peptide release. (The later time point is not known from our experiment, and we only know from another experiment that the final state was seen after 45 s.) Blue: 50S large subunit; orange: 30S small subunit; green: tripeptide; brown: P-tRNA; pink: mRNA; red: compact RF; and blue-purple: extended RF

      Full size image

      Methods

      Components for in vitro translation and fast kinetics

      Buffers and all Escherichia coli (E. coli) components for cell-free protein synthesis were prepared as described22. Ribosomal release complexes (RC) contained tritium (3H) labeled fMet-Phe-Phe-tRNAPhe in the P site and had UAA stop-codon programmed A site. The mRNA sequence used to synthesize the peptide was GGGAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUUAAAUGUUCUUCUAAUGCAGAAAAAAAAAAAAAAAAAAAAA (ORF underlined and bold, SD underlined). Class-1 release factors (RFs), overexpressed in E. coli, had mainly unmethylated glutamine (Q) in the GGQ motif and the RF2 variant contained Ala in position 246. Rate constants for conformational changes of RFs in response to cognate A-site stop codon (kconf) and for ester bond hydrolysis (khydr) at different OH concentrations were estimated as described22. In short, purified release complexes (0.02 µM final concentration) were reacted at 25 °C with saturating amounts of RFs (0.8 µM final) in a quench-flow instrument, and the reaction stopped at different time points by quenching with 17% (final concentration) formic acid. Precipitated [3H]fMet-Phe-Phe-tRNAPhe was separated from the soluble [3H]fMet-Phe-Phe peptide by centrifugation. The amounts of tRNA-bound and free peptides were quantified by scintillation counting of the 3H radiation. Reaction buffer was polymix-HEPES with free Mg2+ concentration adjusted from 5 to 2.5 mM by addition of 2.5 mM Mg2+-chelating UTP. The rate constants for RF association to the A site at 25 °C, ka25, were estimated from their previously published values at 37 °C, ka37 = 60 µM−1 s−1 for RF1 and 23 µM−1 s−1 for RF238 through ka25 = (T2525)·(ŋ37/T37), where T is the absolute temperature and ŋ the water viscosity. Kinetics simulations were carried out with the termination reaction steps modeled as consecutive first-order reactions43.

      Preparation of EM grids and time-resolved cryo-EM

      Quantifoil R1.2/1.3 grids with a 300 mesh size were subjected to glow discharge in H2 and O2 for 25 s using a Solarus 950 plasma cleaning system (Gatan, Pleasanton, CA) set to a power of 10 W. Release complexes and RFs were prepared in the same way as for quench-flow experiments, except the release complexes were unlabeled. For each time point (24 ms and 60 ms), 4 µM of release complexes in polymix-HEPES with 2.5 mM UTP and 6 µM of class-1 release factors in the same buffer were injected into the corresponding microfluidic chip at a rate of 3 µl/s such that they could be mixed and sprayed onto a glow-discharged grid as previously described33. The final concentration of the release complexes and the class-1 release factors after rapid mixing in our microfluidic chip was 2 µM and 3 µM, respectively. As the mixture was sprayed onto the grid, the grid was plunge-frozen in liquid ethane-propane mixture (37%:63%) and stored in liquid nitrogen until it was ready to be imaged.

      Preparation of EM grids and blotting-plunging cryo-EM

      Grids of RC0 and long-incubation complex were prepared with the following protocol. 3 uL sample was applied in the holey grids (gold grids R0.6/1 300 mesh, which was plasma cleaned using the Solarus 950 advanced plasma cleaning system (Gatan, Pleasanton, CA) for 25 s at 10 W using hydrogen and oxygen plasma). Vitrification of samples was performed in a Vitrobot Mark IV (FEI company) at 4 °C and 100% relative humidity by blotting the grids once for 6 s with a blot force 3 before plunging them into the liquid ethane-propane mixture.

      Cryo-EM data collection

      Time-resolved cryo-EM grids were imaged either with a 300 kV Tecnai Polara F30 TEM or a Titan Krios TEM. The images were recorded at a defocus range of −1 to −3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.66 Å or 1.05 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image. Blotting-plunging cryo-EM grids were imaged with a 300 kV Tecnai Polara F30 TEM. The images were recorded at a defocus range of 1–3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.24 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image.

      Cryo-EM data processing

      The beam-induced motion of the sample and the instability of the stage due to thermal drift was corrected using the MotionCor2 software program44. The contrast transfer function (CTF) of each micrograph was estimated using the CTFFIND4 software program45. Imaged particles were picked using the Autopicker algorithm included in the RELION 2.0 software program46. For each time point (Supplementary Figs 6 and  7), 2D classification of the recorded images were used to separate 70S ribosome-like particles from ice-like and/or debris-like particles picked by the Autopicker algorithm and to classify the particles that were picked for further analysis into 70S ribosome-like particle classes. These particle classes were then combined into a single dataset of 70S ribosome-like particles and subjected to a round of 3D classification for the purpose of eliminating those obvious contaminants from the rest of the dataset. This classification was set for 10 classes with the following sampling parameters: Angular sampling interval of 15°, offset search range of 5 pixels and offset search step of 1 pixel. The sampling parameters were progressively narrowed in the course of the 50 classification iterations, down to 3.7° for the angular sampling interval. At the end of the first classification round, two classes were found inconsistent with the known structure of the 70S ribosomes and were thus rejected. The rest of the particles were regrouped together as one class. All particles from this class were re-extracted using unbinned images. A consensus refinement was calculated using these particles. The A site of the 70S ribosome displays fractioned density indicating heterogeneity, then, therefore, the signal subtraction approach was applied. The A-site density was segmented out of the ribosome using Segger in Chimera47. The mass of density identified as release factor was used for creating a mask in RELION with 3 pixels extension and 6 pixels soft edge using relion_mask_create. This mask was used for subtracting the release factor-like signal from the experimental particles. The new particles images were used directly as input in the masked classification run with the number of particles set for ten classes, and with the mask around the release factor-binding region. This run of focused classification resulted in two separate classes, one with compact and one with extended conformation of the release factors. The corresponding raw particles were finally used to calculated consensus refinements. The local resolution of the final maps was computed using ResMap48.

      For the RC0 complex dataset, the software MotionCor244 was used for motion correction and dose weighting. Gctf49 was used for estimation of the contrast transfer function parameters of each micrograph. RELION46 was used for all other image processing steps. Particles picking was done automatically in RELION. Boxed out particles were extracted from dose-weighted micrographs with eight times binning. 2D classifications were initially performed on bin8 particle stacks to remove false positive particles from the particle picking step. 3D classification were performed on bin4 particle stacks. Classes from bin4 and bin2 3D classification showing high-resolution features were saved for further processing steps. Un-binned particles from this class were re-extracted and subjected to auto-refinement. The final density map was sharpened by applying a negative B-factor estimated by automated procedures. Local resolution variations were estimated using ResMap48 and visualized with UCSF Chimera47.

      Model building and refinement

      Models of the E. coli 70S ribosome (5MDV, 5MDW, and 5DFE) were docked into the maps using UCSF Chimera. The pixel size was calibrated by creating the density map from the atomic model and changing the pixel size of the map to maximize the cross-correlation value. For the compact RF1 model, a homology model was generated with the crystal structure of the RF1 (PDB ID: 1ZBT) as a template using the SWISS-MODEL online server50. This homology model was rigid-body-fitted into the map using UCSF Chimera, followed by manual adjustment in Coot51. Due to the lack of density, domain I of RF1 was not modeled.

      Figure preparation

      All figures showing electron densities and atomic models were generated using UCSF Chimera47.

      Data availability

      The data that support the findings of this study are available from the corresponding author upon request. The atomic coordinates and the associated maps have been deposited in the PDB and EMDB with the accession codes 20173, 20184, 20187, 20188, 20193, 20204, 6ORE, 6ORL, 6OSQ, 6OST, 6OT3, and 6OUO. The source data underlying Supplementary Figs 2 and 3 are provided as a Source Data file.

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      Acknowledgements

      This work was supported by HHMI and grants NIH R01 GM55440 and R01 GM29169 (to J.F.), the Swedish Research Council, and the Knut and Alice Wallenberg Foundation (to M.E.), and the Sederholms travel stipend (Uppsala University) (to G.I.).

      Author information

      Author notes
      1. These authors contributed equally: Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar.

      Affiliations

      1. Department of Biochemistry and Molecular Biophysics, Columbia Photo editor download for pc, New York, NY, 10032, USA

        Ziao Fu, Sandip Kaledhonkar, Robert A. Grassucci & Joachim Frank

      2. Department of Cell and Molecular Biology, Uppsala University, Uppsala, 751 24, Sweden

        Gabriele Form z download - Free Activators Ehrenberg

      3. Department of Biological Sciences, Barnard College, New York, NY, 10027, USA

        Binita Shah

      4. Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

        Ming Sun, Bo Chen & Joachim Frank

      Contributions

      Z.F., G.I., S.K., B.S., M.S., B.C., R.A.G., M.E., and J.F. conceived and designed experiments. G.I. carried out biochemical experiments. Z.F., S.K., G.I., and B.S. performed time-resolved cryo-EM experiments. Z.F., S.K., G.I., B.S., and M.S. performed image processing and atomic modeling. Z.F., S.K., G.I., and B.S. analyzed the data. Z.F., G.I., S.K., M.E., and J.F. wrote the manuscript.

      Corresponding author

      Correspondence to Joachim Frank.

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      Competing interests

      The authors declare no competing interests.

      Additional information

      Journal peer review information:Nature Communications thanks Andrei Korostelev, Joseph Puglisi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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      It’s About Time. Crash, Coco, and friends gear up for adventure with a fresh new release across multiple platforms this year.

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      Oct 02, 2020

      Crash Bandicoot™ 4: It’s About Time Available Now

      Join Crash and friends in the direct sequel to the original Crash trilogy, featuring a new art style and a modern take on the classic platforming experience you know and love.

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      Sep 14, 2020

      Crash Bandicoot™ 4: It’s About Time Demo Available This Weekend!

      Pre-Order the game digitally and get access to the Crash Bandicoot™ 4 Demo, available on September 16, 2020*. Read for more information on this demo, as well as Tawna’s return and all new Flashback levels.

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      We're here to help!

      Get answers to frequently asked questions, chat with a support expert, and engage with the support community

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      Our Teams

      • Game Design
      • Art & Animation
      • Brand Management
      • Production
      • Quality Assurance
      • Customer Support
      • Studio Operations
      • Programming
      • Finance & Accounting
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      Источник: https://www.activision.com/

      Free Fire keeps on releasing the advanced servers from time to time. The Latest variant i.e. OB29 was released on 4th August 2021. After OB27, This was the much-waited version by the players. As the new Free Fire server is live now, We are providing you with all the latest form z download - Free Activators related to its VPN, APK file, Download process, Login & registration process with other details like Free Fire Redeem Codes.

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      Before releasing the new update on the global version, Free Fire creates an advanced server and releases the new features there only. This helps the developer to test for the bugs and user experience. Only and number of users get the download the advanced server with the activation code. These users can play and find the bugs to report them back to Garena developers. Such users get free Diamonds as rewards.

      Free Fire OB29 Advance Server (OB29 Update Free Fire)

      OB29 version of Free Fire is out and there will be various features in the game. The key points about FF OB29 are discussed as:

      • The advanced server has various features which are totally free and are not available for everyone. Some of these features are not even released to the public.
      • You will get the chance to hunt for bugs and report them to the Free Fire team. This will earn you free diamonds and other rewards.
      • Online limited Garena FF Advance codes are available so register yourself to get the activation codes.
      • Only Players who have the activation code for the advance server will be able to use it.
      • The advanced server of Free Fire can be installed on any Android smartphone. Soon, the iOS version will be made available.

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      Free Fire Advance Server Registration & Login

      To get the OB29 apk file, the players will have to register to the official Free Fire website. The registrations were opened on 4th August and the last date for the registration is in August 2021. News registrations are closed for now and Free Fire Advance server registrations will open after the release of OB29.

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      The registration can be done only by those who have the FF Advance Server code. Without the code, The users will not be able to make a registration for the new server. The process for FF Advance Server Login & Registration are given below.

      • As of the first step, the user will have to visit the official website of the Free Fire game i.e. ff-advance.ff.garena.com.
      • Now you will see an option to log in using your Facebook account. Use that link and Login to the Free Fire advance server download page.

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      • Now you will be asked to fill in the details like You Full name, Your email address, and your phone number.

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      • Now before opening the downloaded file, the user must enable the “Install from Unknown Source: option in their Android phone settings.
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      Источник: https://www.mpnrc.org/free-fire-advance-server/

      myCSUNsoftware

      What Software is Available?

      Software available to all students:

      • Microsoft Office (can be access from myCSUNsoftware and is available for download)
      • Aleks
      • AMOS
      • ArcGIS 3D (College of S&BS)
      • LibreOffice
      • Mathematica
      • PuTTY
      • R
      • SPSS

      Software available to select disciplines:

      • AutoCAD 3D (Colleges of HHD & ECS)
      • Microsoft Project
      • Microsoft Visio
      • Microsoft Visual Studio
      • Minitab
      • Raven
      • Solidworks (College of ECS)
      • LabVIEW
      • MyITLab
      • Cadence

      myCSUNsoftware 3D Enhancements

      Students in certain classes have access to enhanced versions of applications to utilize 3D functionality: ArcGIS (college of S&BS), AutoCAD (colleges of HHD & ECS), and SolidWorks (college of ECS).

      How do I request additional software?
      To add additional software to myCSUNsoftware, visit myCSUNsoftware - Requesting Software.

      General Access FAQs

      How do I access myCSUNsoftware? 
      Go to mycsunsoftware.csun.edu and sign in with your CSUN user ID and password.

      Can I access myCSUNsoftware from home? 
      Yes. myCSUNsoftware can be accessed at any time, from any location.

      Can I access myCSUNsoftware from a Mac, PC or Linux?
      Yes. myCSUNsoftware can be accessed from any device with an Internet connection. Download the Citrix plug-in client. The system will prompt you for the download if the computer you are using does not have it.

      Can I use my iPad, iPhone, or Android device to access myCSUNsoftware?
      Yes. You will need to download a Citrix Receiver app. For instructions, view the myCSUNsoftware User Guide. 

      Can I access myCSUNsoftware from multiple systems at the same time?
      Yes. You can run myCSUNsoftware on a Windows, Mac and Linux operating system and these can run concurrently.

      Can I print within the application?
      Yes. Printing within the virtual application is the same as printing in any other application locally installed on your system.

      Can I access the myCSUNsoftware storage drive when I’m not in the myCSUNsoftware environment?
      Yes. The myCSUNsoftware storage drive can be accessed outside of the myCSUNsoftware environment so you have access to your files. Accessing these files is done by mapping to the myCSUNsoftware storage much like you do for myCSUNbox. For instructions, view the myCSUNsoftware User Guide.

      Will my sessions time out if there is inactivity?
      When using myCSUNsoftware, be aware that there are two different timeouts; one is the webpage timeout and the other is the program or application timeout. Without activity, the webpage times out after 15 minutes and will prompt you to log back in. Even if the webpage times out, the application will continue to run for up to 90 minutes without any activity, and then it will close. Within that 90 minute period, you can log in to the webpage, launch the same applications and you will still be able to save your work. If the application is inactive for 120 minutes, it will disconnect and work will be lost. Remember to save your work often.  

      Is myCSUNsoftware ever unavailable?
      To keep myCSUNsoftware running optimally, scheduled maintenance takes place on Saturdays from midnight to 6 a.m. and access may be intermittent during this time. 

      Microsoft Office FAQs

      How many copies of Microsoft Office can I download?
      You can install Microsoft Office on up to 5 personal computers. 

      Can I install Microsoft Office on a Mac?
      Yes

      What versions of Office are available?
      Office 2016

      Technical FAQs

      What do I do when I see a white screen, java error or .ica error once I attempt to launch software after I log in?
      Log in to myCSUNsoftware. From the main menu, select the Settings link located in the grey bar at top right. In the Settings window under the General heading, select the Run Client Detection link. Next, when prompted, install the Citrix client plug-in, close the browser, save all work and restart your computer. Log in to myCSUNsoftware, return to the Settings window under the General heading and confirm that it reads, The Native Client is currently selected. You should now be ready to launch software in myCSUNsoftware.

      When I try to launch an application, how do I get past the prompt that tells me to save or open an .ica file, even though I am I logged in and have the Citrix client plug-in installed?
      The Citrix client plug-in may have been downloaded to your computer, but not installed. Find the Downloaded file and Run the installation process. You may need to re-download the plug-in. If so, log in to myCSUNsoftware, choose settings and select native client check. Follow the prompts to re-download the client, find the file downloaded, and run through the installation process. Once complete, re-visit the log in page and you should be able to access applications.

      Why can’t I print?
      You may be using a printer that cannot be detected or has an outdated printer driver. 

      Where do I save my work? 
      myCSUNsoftware provides a high speed storage option. The recommended storage option is the network share drive: ZDrive or (Z:). The ZDrive is also identified with a short description such as \\vslfiles. You also have the ability to save to your local computer, portable flash drive or myCSUNbox.

      When I’m using the virtual application, how can I see my computer drives such as C, F, etc. to locate my files?
      Check the Citrix profile settings:

      • On a Mac: Select System Preferences (the Apple menu). In the Other heading, select the Citrix Online Plug-in menu. From the Citrix Online Plug-in dialog box, select the Devices tab. Under the Mapped Drives section, select the drive you are accessing and change the Read and Write columns to Always or Ask me first.
      • On a PC: With the myCSUNsoftware program running, select the Citrix iconin the bottom right corner of your Windows Task Bar. The Citrix Connection Center dialog box opens. Under the Session Security section, change the Files field and USB/Other Devices field to Always or Ask Permission.

      Is there something I can do if myCSUNsoftware is running slowly?
      To improve the performance, check the following:

      • Ensure you are accessing your saved file from the myCSUNsoftware high speed file storage. If your saved file resides in a place other than the myCSUNsoftware high speed file storage, try saving your file to the myCSUNsoftware storage and open it within the myCSUNsoftware environment. (Indicate that it is the Z: or it will be labeled as ‘vsfiles’) 
      • Verify the Citrix client plug-in is installed and running. 
      • Verify your Internet connection is DSL or greater. The recommended minimum speed for accessing myCSUNsoftware is 100kbps or .1mbps. To check your connection speed, go to http://www.speakeasy.net/speedtest/.
      Источник: https://www.csun.edu/it/mycsunsoftware-0

      Specifies an address to listen on for a stream (), datagram (), or sequential packet () socket, respectively. The address can be written in various formats:

      If the address starts with a slash (""), it is read as file system socket in the socket family.

      If the address starts with an at symbol (""), it is read as abstract namespace socket in the family. The "" is replaced with a character before binding. For details, see unix(7).

      If the address string is a single number, it is read as port number to listen on via IPv6. Depending on the value of (see below) this might result in the service being available via both IPv6 and IPv4 (default) or just via IPv6.

      If the address string is a string in the format "", it is interpreted as IPv4 address and port .

      If the address string is a string in the format "", it is interpreted as IPv6 address and port. An optional interface scope (interface name or number) may be specified after a "" symbol: "". Interface scopes are only useful with link-local addresses, because the kernel ignores them in other cases. Note that if an address is specified as IPv6, it might still make the service available via IPv4 too, depending on the setting (see below).

      If the address string is a string in the format "", it is read as CID on a port address in the templatetoaster 7 patch family. The CID is a unique 32-bit integer identifier in analogous to an IP address. Specifying the CID is optional, and may be set to the empty string.

      Note that (i.e. ) is only available for sockets. (i.e. ) when used for IP sockets refers to TCP sockets, (i.e. ) to UDP.

      These options may be specified more than once, in which case incoming traffic on any of the sockets will trigger service activation, and all listed sockets will be passed to the service, regardless of whether there is incoming traffic on them or not. If the empty string is assigned to any of these options, the list of addresses to listen on is reset, all prior uses of any of these options will have no effect.

      It is also possible to have more than one socket unit for the same service when using and the service will receive all the sockets configured in all the socket units. Sockets configured in one unit are passed in the order of configuration, but no ordering between socket units is specified.

      If an IP address is used here, it is often desirable to listen on it before the interface it is configured on is up and running, and even regardless of whether it will be up and running at any point. To deal with this, it is recommended to set the option described below.

      Specifies a file system FIFO (see fifo(7) for details) to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above.

      Specifies a special file in the file system to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above. Use this to open character device nodes as well as special files in and .

      Specifies a Netlink family to create a socket for to listen on. This expects a short string referring to the family name (such as or ) as argument, optionally suffixed by a whitespace followed by a multicast group integer. Behavior otherwise is very similar to the directive above.

      Specifies a POSIX message queue name to listen on (see mq_overview(7) for details). This expects a valid message queue name (i.e. beginning with ""). Behavior otherwise is very similar to the directive above. On Linux message queue descriptors are actually file descriptors and can be inherited between processes.

      Specifies a USB FunctionFS endpoints location to listen on, for implementation of USB gadget functions. This expects an absolute file system path of a FunctionFS mount point as the argument. Behavior otherwise is very similar to the directive above. Use this to open the FunctionFS endpoint . When using this option, the activated service has to have the and options set.

      Takes one of or. The socket will use the UDP-Lite () or SCTP () protocol, respectively.

      Takes one of or. Controls the IPV6_V6ONLY socket option (see ipv6(7) for details). IfIPv6 sockets bound will be accessible via both IPv4 and IPv6. If they will be accessible via IPv6 only. If (which is the default, surprise!), the system wide default setting is used, as controlled by which in turn defaults to the equivalent of .

      Takes an unsigned integer argument. Specifies the number of connections to queue that have not been accepted yet. This setting matters only for stream and sequential packet sockets. See listen(2) for details. Defaults to SOMAXCONN (128).

      Specifies a network interface name to bind this socket to. If set, traffic will only be accepted from the specified network interfaces. This controls the socket option (see socket(7) for details). If this option is used, an implicit dependency from this socket unit on the network interface device unit is created (see systemd.device(5)). Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

      , ¶

      Takes a UNIX user/group name. When specified, all sockets and FIFO nodes in the file system are owned by the specified user and group. If unset (the default), the nodes are owned by the root user/group (if run in system context) or the invoking user/group (if run in user context). If only a user is specified but no group, then the group is derived from the user's default group.

      If listening on a file system socket or FIFO, this option specifies the file system access mode used when creating the file node. Takes an access mode in octal notation. Defaults to 0666.

      If listening on a file system socket or FIFO, the parent directories are automatically created if needed. This option specifies the file system access mode used when creating these directories. Takes an access mode in octal notation. Defaults to 0755.

      Takes a boolean argument. If yes, a service instance is spawned for each incoming connection and only the connection socket is passed to it. If no, all listening sockets themselves are passed to the started service unit, and only one service unit is spawned for all connections (also see above). This value is ignored for datagram sockets and FIFOs where a single service unit unconditionally handles all incoming traffic. Defaults to. For performance reasons, it is recommended to write new daemons only in a way that is suitable for . A daemon listening on an socket may, but does not need to, call close(2) on the received socket before exiting. However, it must not unlink the socket from a file system. It should not invoke shutdown(2) on sockets it got withbut it may do so for sockets it got with set. Setting is mostly useful to allow daemons designed for usage with inetd(8) to work unmodified with systemd socket activation.

      For IPv4 and IPv6 connections, the environment variable will contain the remote IP address, and will contain the remote port. This is the same as the format used by CGI. Forthe port is the IP protocol.

      Takes a boolean argument. May only be used in conjunction with. If true, the specified special file is opened in read-write mode, if false, in read-only mode. Defaults to false.

      Takes a boolean argument. May only be used when . If yes, the socket's buffers are cleared after the triggered service exited. This causes any pending data to be flushed and any pending incoming connections to be rejected. If no, the socket's buffers won't be cleared, permitting the service to handle any pending connections after restart, which is the usually expected behaviour. Defaults to.

      The maximum number of connections to simultaneously run services instances for, when is set. If more concurrent connections are coming in, they will be refused until at least one existing connection is terminated. This setting has no effect on sockets configured with or datagram sockets. Defaults to 64.

      The maximum number of connections for a service per source IP address. This is very similar to the directive above. Disabled by default.

      Takes a boolean argument. If true, the TCP/IP stack will send a keep alive message after 2h (depending on the configuration of ) for all TCP streams accepted on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults to .

      Takes time (in seconds) as argument. The connection needs to remain idle before TCP starts sending keepalive probes. This controls the TCP_KEEPIDLE socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 7200 seconds (2 hours).

      Takes time (in seconds) as argument between individual keepalive probes, if the socket option has been set on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 75 seconds.

      Takes an integer as argument. It is the number of form z download - Free Activators unacknowledged probes to send before considering the connection dead and notifying the application layer. This controls the TCP_KEEPCNT socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is form z download - Free Activators 9.

      Takes a boolean argument. TCP Nagle's algorithm works by combining a number of small outgoing messages, and sending them all at once. This controls the TCP_NODELAY socket option (see tcp(7)). Defaults to .

      Takes an integer argument controlling the priority for all traffic sent from this socket. This controls the socket option (see socket(7) for details.).

      Takes time (in seconds) as argument. If set, the listening process will be awakened only when data arrives on the socket, and not immediately when connection is established. When this option is set, the socket option will be used (see tcp(7)), and the kernel will ignore initial ACK packets without any data. The argument specifies the approximate amount of time the kernel should wait for incoming data before falling back to the normal behavior of honoring empty ACK packets. This option is beneficial for protocols where the client sends the data first (e.g. HTTP, in contrast to SMTP), because the server process will not be woken up unnecessarily before it can take any action.

      If the client also uses the option, the latency of the initial connection may be reduced, because the kernel will send data in the final packet establishing form z download - Free Activators connection (the third packet in the "three-way handshake").

      Disabled by default.

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      Takes an integer argument controlling the receive or send buffer sizes of this socket, respectively. This controls the and socket options (see socket(7) for details.). The usual suffixes K, M, G are supported and are understood to the base of 1024.

      Takes an integer argument controlling the IP Type-Of-Service field for packets generated from this socket. This controls the socket option (see ip(7) for jpg to pdf converter full crack - Crack Key For U details.). Either a numeric string or one of, or may be specified.

      Takes an integer argument controlling the IPv4 Time-To-Live/IPv6 Hop-Count field for packets generated from this socket. Coreldraw x9 crack only sets the / socket options (see ip(7) and ipv6(7) for details.)

      Takes an integer value. Controls the firewall mark of packets generated by this socket. This can be used in the firewall logic to filter packets from this socket. This sets the socket option. See iptables(8) for details.

      Takes a boolean value. If true, allows multiple bind(2)s to this TCP or UDP port. This controls the socket option. See socket(7) for details.

      Takes a string value. Controls the extended form z download - Free Activators attributes "", "" and "", respectively, i.e. the security label of the FIFO, or the security label for the incoming or outgoing connections of the socket, respectively. See Smack.txt for details.

      Takes a boolean argument. When true, systemd will attempt to figure out the SELinux label used for the instantiated service from the information handed by the peer over the network. Note that only the security level is used from the information provided by the peer. Other parts of the resulting SELinux context originate from either the target xara web designer premium review - Crack Key For U binary that is effectively triggered by socket unit or from the value of the option. This configuration option applies only when activated service is passed in single socket file descriptor, i.e. service instances that have standard input connected to a socket or services triggered by exactly one socket unit. Also note that this option is useful only when MLS/MCS SELinux policy is deployed. Defaults to "".

      Takes a size in bytes. Controls the pipe buffer size of FIFOs configured in this socket unit. See fcntl(2) for details. The usual suffixes K, M, G are supported and are understood to the base of 1024.

      , ¶

      These two settings take integer values and control the mq_maxmsg field or the mq_msgsize field, respectively, when creating the message queue. Note that either none or both of these variables need to be set. See mq_setattr(3) for details.

      Takes a boolean value. Controls whether the socket can be bound to non-local IP addresses. This is useful to configure sockets listening on specific IP addresses before those IP addresses are successfully configured on a network interface. This sets the / socket option. For robustness reasons it is recommended to use this option whenever you bind a socket to a specific IP address. Defaults to .

      Takes a boolean value. Controls the / socket option. Defaults to .

      Takes a boolean value. This controls the socket option, which allows broadcast datagrams to be sent from this socket. Defaults to .

      Takes a boolean value. This controls the socket option, which allows sockets to receive the credentials of the sending process in an ancillary message. Defaults to .

      Takes a boolean value. This controls the socket option, which allows sockets to receive the security context of the sending process in an ancillary message. Defaults to .

      Takes a boolean value. This controls the or socket options, which enable reception of additional per-packet metadata as ancillary message, on, and sockets. Defaults to .

      Takes one of "", "" (alias: "", "") or "" (alias: ""). This controls the or form z download - Free Activators options, and enables whether ingress network traffic shall carry timestamping metadata. Defaults to .

      Takes a string value. Controls the TCP congestion algorithm used by this socket. Should be one of "", "", "", "" or any other available algorithm supported by the IP stack. This setting applies only to stream sockets.

      , ¶

      Takes one or more command lines, which are executed before or after the listening sockets/FIFOs are created and bound, respectively. The first token of the command line must be an absolute filename, then followed by arguments for the process. Multiple command lines may be specified following the same scheme as used for of service unit files.

      , ¶

      Additional commands that are executed before or after the listening sockets/FIFOs are closed and removed, respectively. Multiple command lines may be specified following the same scheme as used for of service unit files.

      Configures the time to wait for the commands specified in and to finish. If a command does not exit within the configured time, the socket will be considered failed and be shut down again. All commands still running will be terminated forcibly via and after another delay of this time with. (See in systemd.kill(5).) Takes a unit-less value in seconds, or a time span value such as "5min 20s". Pass "" to disable the timeout logic. Defaults to from the manager configuration file (see systemd-system.conf(5)).

      Specifies the service unit name to activate on incoming traffic. This setting is only allowed for sockets with. It defaults to the service that bears the same name as the socket (with the suffix replaced). In most cases, it should not be necessary to use this option. Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

      Takes a boolean argument. If enabled, any file nodes created by this socket unit are removed when it is stopped. This applies to sockets in the file system, POSIX message queues, FIFOs, as well as any symlinks to them configured with . Normally, it should not be necessary to use this option, and is not recommended as services might continue to run after the socket unit has been terminated and it should still be possible to communicate with them via their file system node. Defaults to off.

      Takes a list of file system paths. The specified paths will be created as symlinks to the socket path or FIFO path of this socket unit. If this setting is used, only one socket in the file system or one FIFO may be configured for the socket unit. Use this option to manage one or more symlinked alias names for a socket, binding their lifecycle together. Note that if creation of a symlink fails this is not considered fatal for the socket unit, and the socket unit may still start. If an empty string is assigned, the list of paths is reset. Defaults to an empty list.

      Assigns a name to all file descriptors this socket unit encapsulates. This is useful to help activated services identify specific file descriptors, if multiple fds are passed. Services may use the sd_listen_fds_with_names(3) call to acquire the names configured for the received file descriptors. Names may contain any ASCII character, but must exclude control characters and "", and must be at most 255 characters in length. If this setting is not used, the file descriptor name defaults to the name of the socket unit, including its suffix.

      , ¶

      Configures a limit on how often this socket unit my be activated within a specific time interval. The may be used to configure the length of the time interval in the usual time units "", "", "", "", "", … and defaults to 2s (See systemd.time(7) for details on the various time units understood). The setting takes a positive integer value and specifies the number of permitted activations per time interval, and defaults to 200 for sockets (thus by default permitting 200 activations per 2s), and 20 otherwise (20 activations per 2s). Set either to 0 to disable any form of trigger rate limiting. If the limit is hit, the socket unit is placed into a failure mode, and will not be connectible anymore until restarted. Note that this limit is enforced before the service activation is enqueued.

      Источник: https://www.freedesktop.org/software/systemd/man/systemd.socket.html

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      Capture your vision

      The multi-award winning Atomos Ninja V Monitor-Recorder features a stunningly bright 5.2” 10-bit HDR display with 1000nits of output. The sleek aluminium body is crafted for maximum durability, weighing in at just 360g and measuring only 25mm thick – easily attaching to any camera rig.

      Ninja V records directly from your camera’s sensor to a wide range of codecs: Apple ProRes, ProRes RAW, Avid DNx and H.265 (HEVC)*.This provides wide compatibility across camera types and edit software, giving you a new degree of creative control long before you press record.

      * H.265 is an optional paid activation via my.atomos.com

      The Ninja V takes all your creativity and brings it to life in the video you capture, regardless of whether you’re shooting cinematic productions, or vlogs for your social media channel. Whether you shoot HDR or regular SDR. Whether you use a professional cinema camera or a prosumer mirrorless camera.

      Ninja V continues to innovate with regular free firmware updates, widely supported ProRes RAW recording, SDI connectivity with AtomX accessories and professional feature activations including H.265 recording.

      Control what you see from Capture to SDR/HDR Delivery

        RECORDING

        Ninja V can record using the 10-bit H.265 (HEVC) codec by purchasing the upgrade at my.atomos.com for $99 (USD)

        H.265 adds even more versatility to the Ninja V, by adding a high-quality compressed codec option for recording. H.265 codec is used for the final delivery of high quality video projects with very small file sizes. HEVC was specifically created to provide support for UHD HDR deliverables with wide color gamuts, and enables you to easily share and allow play back of HDR content on a wide range of devices.

        If you’d like to know more about H.265 and Ninja V you can continue reading here, learn more about Ninja V, read more below or purchase now at my.atomos.com

        Read more

        MY.ATOMOS.COM

        The latest developments for Ninja V include

        • Multicam Switching

          with AtomX CAST

        • 5.9K ProRes RAW

          with selected cameras

        HDR / SDR

        State-of-the-art content creation

        Ninja V is the ultimate field monitor for the on-the-go film and video creator that values capturing to high quality industry standard codecs for SDR, HDR and next generation content delivery. The Ninja V’s state-of-the-art screen maintains clarity and color in the brightest of conditions and displays the widest dynamic range in HDR.

        The unique AtomHDR engine processes the incoming Log/RAW/HDR signals from your camera or device and displays in all its HDR glory on the Ninja V screen. There are matching profiles for a plethora of cameras, with more added as they emerge. Prefer to work in Rec.709? The Ninja V has you covered with the ability to load custom LUTs to match your workflow.

        UNLOCK PRO FEATURES


        RAW Recording
        from SDI Cameras

        Ninja V records ProRes RAW over SDI with compatible cameras using the AtomX SDI Module and paid upgrade of $99. The Activation Key for SDI RAW and HDMI RAW to SDI Video can be be purchased via your my.atomos.com account.

        These features bring even more versatility to the Ninja V, providing professional I/O options for a multitude of cameras – from entry level mirrorless cameras through to the latest compact cinema and high-end professional cameras. Read more about how Ninja V can expand your RAW eco-system.

        Ninja V Pro Kit

         

        MY.ATOMOS.COM
        <br/>RAW Recording<br>from SDI Cameras

        Instant Playback & Review

        Ninja V takes exactly what your camera sensor is capturing, enhances it and allows you to view and record it in amazing detail. Ninja V gives you the ability to record and instantly playback, review and tag your favourites takes to create playlists. This portable powerhouse gives you complete confidence in what you’re capturing, so you’re free to push creative boundaries more than ever before.

         

        Instant Playback & Review
        RECORD RAW

        Ninja V & ProRes RAW

        The number of cameras which now support RAW recording over HDMI on the Ninja V continues to grow. ProRes RAW combines the visual and workflow benefits of RAW video with the incredible real-time performance of ProRes. This format gives filmmakers enormous latitude when adjusting the look of their images, making it ideal for HDR workflows.

        COMPATIBLE CAMERAS
        Ninja V & ProRes RAW

        ProRes RAW Recording with Ninja V

        Learn more about Ninja V and the latest cameras at Atomos Academy

        Optional accessories for your Ninja V

        Essential accessories for capture, post-production and streaming

        Optional accessories for your Ninja V
        • AtomX SDI

          Input 12G/6G/3G/HD with the ability to toggle between input channel sources on SDI 1 / 2. Supporting up to 4kp60. Dual-link support for 1.5G/3G and 6G.

        • Battery Eliminator

          Power select Atomos monitor/recorder units

        • Sunhood

          Block excess reflections from washing out the displayed image

        • SSD

          Custom made for the new Atomos Ninja V monitor recorder

        • Docking Station

          Offload the drive contents direct to your computer

        • Connect

          Bridge recording and monitoring with streaming platforms such as YouTube, Twitch and OBS.

        • Monitor Mount

          Features a quick release plate, as well as 360° pan and 180° tilt functionality

        • DTap Cable

          D-Tap to DC Locked connector Barrel Coiled Cable for Battery Eliminator

        • AtomX Arm

          Attach your Atomos monitor to your camera cage or rig via a quick release workflow.

        PRO MONITORING

        Access All Areas

        The Ninja V offers a huge range of features that can enhance your workflow, from AtomOS Monitor Assist features like Focus Peaking, Zoom, VectorscopesFalse Color through to Safe Areas, Cine Guides and our AtomHDR processing engine that allows realtime Log>PQ/HLG previewing and more. Customizable settings & view modes — and instantly clear all overlays with a simple touch of the screen. Take control of your production at capture with Atomos Ninja V.

        Focus Peaking
        Customizable

        Weddings. Parties. Anything.

        Universally popular, Ninja V ticks more boxes for more filmmakers than any other 5” monitor on the market. And it will only get better. The Ninja V’s features continue to improve and evolve with every new AtomOS release. Not only will additional ProRes RAW capable cameras be supported, other new features are constantly being added for free.

        Recent additions include a wider range of frame guides (1:1, 4:5, 1:91:1 and 9:16) – perfect for social media content creation. A nine-grid overlay makes composing using the rule of thirds easy. The addition of an ARRI style false colour scale bolsters Ninja V’s impressive professional credentials.

        Weddings. Parties. Anything.

        Lock the shot with Ninja V’s monitoring and composition essentials.

        • Social Guides

          1:1 / 4:5 / 1:91:1 / 9:16

        • Safe Areas

          TV / Screen Guides

        • Frame Guides

          SMPTE standards

        Learn, Inspire, Create.

         

        Check out the latest content created using Atomos Ninja V.
        Features from Creators, tips from Pros and the latest innovations from Atomos.

        BROWSE ACADEMY
        SSD MEDIA

        Break through the recording time barrier

        Shooting 4K and above in advanced formats like ProRes, DNx and ProRes RAW requires storage media with ample volume, fast transfer times and high, sustained read and write speeds.

        AtomX SSDmini drives complete the Ninja V’s digital workflow. Smaller than conventional SATA SSDs they are an affordable professional alternative to recording to a camera’s internal memory cards. Offering up to 2TB storage, a sequential read speed of up to 550MB/s and write speed up to 500MB/s, these drives can record up to 150 minutes of 4K ProRes on a single drive. Measuring 8cm long, 7.5cm wide and weighing as little as 88g, the custom built drives neatly fit the Ninja V’s compact proportions.

        Break through the recording time barrier

        Fast. Reliable. Compact. Rugged.

          ProRes RAW

          Olympus E-M1X & E-M1 MIII with the Ninja V

          WATCH: Janne Amunét Director from Kauas Creative speaks first-hand of his experience shooting in ProRes RAW on the Olympus E-M1X & E-M1 MIII with the Ninja V. Understand the key features that make up the Olympus E-M1X & E-M1 MIII such as weatherproofing and image stabilisation and what this means when the mirrorless cameras are paired with the Ninja V to shoot in ProRes RAW.

           

          Related Posts

          FEATURE

          ProRes RAW on Z CAM + Ninja V

          WATCH: Go behind the scenes of UN/SEEN, an artistic abstract short film by filmmaker/director James Tonkin. UN/SEEN was shot on the Z CAM E2-F6 full-frame cinema camera and the Atomos Ninja V HDR monitor-recorder in Apple ProRes RAW.

          The behind the scenes video was shot on the Z CAM E2 and Ninja V in ProRes RAW. Watch the finished video below. Then view final project inHDR.

           

          ATOMOS ACADEMY

          ATOMX SYNC

          Making multi-camera mainstream

          You no longer need a huge budget or a highly skilled editor to produce perfectly synchronised, multicamera video. The optional AtomX SYNC module sits on the back of the Ninja V, integrating it into a Timecode Systems powered multi-camera sync network. The advanced long-range RF system is completely wireless, offering incredible accuracy and stability. Effortlessly synchronise your recordings with multiple camera and sound sources. With frame-accurate timecode embedded directly into each recording you can accurately edit together multiple video and audio sources effortlessly using automated functions in all popular editing software.

          LEARN MORE

          CALIBRATION

          Trust what you see

          Ninja V is your go anywhere monitoring system. Any camera, anywhere, any time of day, you can trust that you’re accurately seeing what you are recording. To ensure reliable and dependable accuracy, Atomos have partnered with calibration leader X-Rite to ensure all of our monitors operate seamlessly across Log/HDR capture, Post Production and HRD/SDR delivery. Ninja V is easy to calibrate, ensuring you are seeing your images accurately.

           

          X-RITE i1

          Unlock the full potential of your camera

          The diversity of codecs makes the Ninja V compatible with all major editing software packages. In terms of capture, virtually any HDMI or SDI source is supported, including cameras from Nikon, FujiFilm, Canon, Panasonic, Sony, Z-Cam, Olympus, RED and ARRI.

          Class leading monitoring technology

          • 60-240hz

            Recording frequency

          • Auto HDR

            Flags for TV setup

          Class leading monitoring technology
          • Zoom

          • Focus Peaking

          • LUTS

          • Zebra

          Gaming

          Record 4K HDR Gaming

          Ninja V is a stand-alone system for 4k UHD, HDR and high frame rate capture that eliminates expensive, complicated and unreliable PC setups. Ninja V enables simple recording, monitoring and instant review. Capture every detail in HDR and automatically include all the correct HDR flags ready for upload to YouTube. Ideal for games development testing, pre-release capture sessions or just to show off your skills!

          Ninja V

          Universally popular, Ninja V ticks more boxes for more filmmakers than any other 5” monitor on the market.

          $

          Please note:
          Specifications are subject to change without notice.
          All information correct at time of publishing.

          ° Paid activation. Available at myatomos.com

          FIND A RESELLER
          Ninja V

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          These Atomos product provide similar features. Try the COMPARE PRODUCTS feature to find out more.

          Spark a creative journey.
          Join the Atomos Community.

          Источник: https://www.atomos.com/products/ninja-v

          Z-DNA

          Z-DNA is one of the many possible double helical structures of DNA. It is a left-handed double helical structure in which the helix winds to the left in a zigzag pattern, instead of to the right, like the more common B-DNA form. Z-DNA is thought to be one of three biologically active double-helical structures along with A-DNA and B-DNA.

          History[edit]

          Left-handed DNA was first discovered by Robert Wells and colleagues, during their studies of a repeating polymer of inosine–cytosine.[1] They observed a "reverse" circular dichroism spectrum for such DNAs, and interpreted this (correctly) to mean that the strands wrapped around one another in a left-handed fashion. The relationship between Z-DNA and the more familiar B-DNA was indicated by the work of Pohl and Jovin,[2] who showed that the ultraviolet circular dichroism of poly(dG-dC) was nearly inverted in 4 Msodium chloride solution. The suspicion that this was the result of a conversion from B-DNA to Z-DNA was confirmed by examining the Raman spectra of these solutions and the Z-DNA crystals.[3] Subsequently, a crystal structure of "Z-DNA" was published which turned out to be the first single-crystal X-ray structure of a DNA fragment (a self-complementary DNA hexamer d(CG)3). It was resolved as a left-handed double helix with two antiparallel chains that were held together by Watson–Crick base pairs (see X-ray crystallography). It was solved by Andrew H. J. Wang, Alexander Rich, and coworkers in 1979 at MIT.[4] The crystallisation of a B- to Z-DNA junction in 2005[5] provided a better understanding of the potential role Z-DNA plays in cells. Whenever a segment of Z-DNA forms, there must be B–Z junctions at its two ends, interfacing it to the B-form of DNA found in the rest of the genome.

          In 2007, the RNA version of Z-DNA, Z-RNA, was described as a transformed version of an A-RNA double helix into a left-handed helix.[6] The transition from A-RNA to Z-RNA, however, was already described in 1984.[7]

          Structure[edit]

          B–Z junction bound to a Z-DNA binding domain. Note the two highlighted extruded bases. From PDB: 2ACJ​.

          Z-DNA is quite different from the right-handed forms. In fact, Z-DNA is often compared against B-DNA in order to illustrate the major differences. The Z-DNA helix is left-handed and has a structure that repeats every other base pair. The major and minor grooves, unlike A- and B-DNA, show little difference in width. Formation of this structure is generally unfavourable, although certain conditions can promote it; such as alternating purine–pyrimidine sequence (especially poly(dGC)2), negative DNA supercoiling or high salt and some cations (all at physiological temperature, 37 °C, and pH 7.3–7.4). Z-DNA can form a junction with B-DNA (called a "B-to-Z junction box") in a structure which involves the extrusion of a base pair.[8] The Z-DNA conformation has been difficult to study because it does not exist as a stable feature of the double helix. Instead, it is a transient structure that is occasionally induced by biological activity and then quickly disappears.[9]

          Predicting Z-DNA structure[edit]

          It is possible to predict the likelihood of a DNA sequence forming a Z-DNA structure. An algorithm for predicting the propensity of DNA to flip from the B-form to the Z-form, ZHunt, was written by P. Shing Ho in 1984 at MIT.[10] This algorithm was later developed by Tracy Camp, P. Christoph Champ, Sandor Maurice, and Jeffrey M. Vargason for genome-wide mapping of Z-DNA (with Ho as the principal investigator).[11]

          Pathway of formation of Z-DNA from B-DNA[edit]

          Since the discovery and crystallization of Z-DNA in 1979, the configuration has left scientists puzzled about the pathway and mechanism from the B-DNA configuration to the Z-DNA configuration.[12] The conformational change from B-DNA to the Z-DNA structure was unknown at the atomic level, but in 2010, computer simulations conducted by Lee et al. were able to computationally determine that the step-wise propagation of a B-to-Z transition would provide a lower energy barrier than the previously hypothesized concerted mechanism.[13] Since this was computationally proven, the pathway would still need to be tested experimentally in the lab for further confirmation and validity, in which Lee et al. specifically states in their journal article, "The current [computational] result could be tested by Single-molecule FRET (smFRET) experiments in the future."[13] In 2018, the pathway from B-DNA to Z-DNA was experimentally proven using smFRET assays.[14] This was performed by measuring the intensity values between the donor and acceptor fluorescent dyes, also known as Fluorophores, in relation to each other as they exchange electrons, while tagged onto a DNA molecule.[15][16] The distances between the fluorophores could be used to quantitatively calculate the changes in proximity of the dyes and conformational changes in the DNA. A Z-DNA high affinity binding protein, hZαADAR1,[17] was used at varying concentrations to induce the transformation from B-DNA to Z-DNA.[14] The smFRET assays revealed a B* transition state, which formed as the binding of hZαADAR1 accumulated on the B-DNA structure and stabilized it.[14] This step occurs to avoid high junction energy, in which the B-DNA structure is allowed to undergo a conformational change to the Z-DNA structure without a major, disruptive change in energy. This result coincides with the computational results of Lee et al. proving the mechanism to be step-wise and its purpose being that it provides a lower energy barrier for the conformational change from the B-DNA to Z-DNA configuration.[13] Contrary to the previous notion, the binding proteins do not actually stabilize the Z-DNA conformation after it is formed, but instead they actually promote the formation of the Z-DNA directly from the B* conformation, which is formed by the B-DNA structure being bound by high affinity proteins.[14]

          Biological significance[edit]

          A biological role for Z-DNA in the regulation of type I interferon responses has been confirmed in studies of three well-characterized rare Mendelian Diseases: Dyschromatosis Symmetrica Hereditaria (OMIM: 127400), Aicardi-Goutières syndrome (OMIM: 615010) and Bilateral Striatal Necrosis/Dystonia. Families with haploid ADAR transcriptome enabled mapping of Zα variants directly to disease, showing that genetic information is encoded in DNA by both shape and sequence.[18] A role in regulating type I interferon responses in cancer is also supported by findings that 40% of a panel of tumors were dependent on the ADAR enzyme for survival.[19]

          In previous studies, Z-DNA was linked to both Alzheimer's disease and systemic lupus erythematosus. To showcase this, a study was conducted on the DNA found in the hippocampus of brains that were normal, moderately affected with Alzheimer's disease, and severely affected with Alzheimer's disease. Through the use of circular dichroism, this study showed the presence of Z-DNA in the DNA of those severely affected.[20] In this study it was also found that major portions of the moderately affected DNA was in the B-Z intermediate conformation. This is significant because from these findings it was concluded that the transition from B-DNA to Z-DNA is dependent on the progression of Alzheimer's disease.[20] Additionally, Z-DNA is associated with systemic lupus erythematosus (SLE) through the presence of naturally occurring antibodies. Significant amounts of anti Z-DNA antibodies were found in SLE patients and were not present in other rheumatic diseases.[21] There are two types of these antibodies. Through radioimmunoassay, it was found that one interacts with the bases exposed on the surface of Z-DNA and denatured DNA, while the other exclusively interacts with the zig-zag backbone of only Z-DNA. Similar to that found in Alzheimer's disease, the antibodies vary depending on the stage of the disease, with maximal antibodies in the most active stages of SLE.

          Z-DNA in transcription[edit]

          Z-DNA is commonly believed to provide torsional strain relief during transcription, and it is associated with negative supercoiling.[5][22] However, while supercoiling is associated with both DNA transcription and replication, Z-DNA formation is primarily linked to the rate of transcription.[23]

          A study of human chromosome 22 showed a correlation between Z-DNA forming regions and promoter regions for nuclear factor I. This suggests that transcription in some human genes may be regulated by Z-DNA formation and nuclear factor I activation.[11]

          Z-DNA sequences downstream of promoter regions have been shown to stimulate transcription. The greatest increase in activity is observed when the Z-DNA sequence is placed three helical turns after the promoter sequence. Furthermore, Z-DNA is unlikely to form nucleosomes, which are often located after a Z-DNA forming sequence. Because of this property, Z-DNA is hypothesized to code for nucleosome positioning. Since the placement of nucleosomes influences the binding of transcription factors, Z-DNA is thought to regulate the rate of transcription.[24]

          Developed behind the pathway of RNA polymerase through negative supercoiling, Z-DNA formed via active transcription has been shown to increase genetic instability, creating a propensity towards mutagenesis near promoters.[25] A study on Escherichia coli found that gene deletions spontaneously occur in plasmid regions containing Z-DNA-forming sequences.[26] In mammalian cells, the presence of such sequences was found to produce large genomic fragment deletions due to chromosomal double-strand breaks. Both of these genetic modifications have been linked to the gene translocations found in cancers such as leukemia and lymphoma, since breakage regions in tumor cells have been plotted around Z-DNA-forming sequences.[25] However, the smaller deletions in bacterial plasmids have been associated with replication slippage, while the larger deletions associated with mammalian cells are caused by non-homologous end-joining repair, which is known to be prone to error.[25][26]

          The toxic effect of ethidium bromide (EtBr) on trypanosomas is caused by shift of their kinetoplastid DNA to Z-form. The shift is caused by intercalation of EtBr and subsequent loosening of DNA structure that leads to unwinding of DNA, shift to Z-form and inhibition of DNA replication.[27]

          Discovery of the Zα domain[edit]

          The first domain to bind Z-DNA with high affinity was discovered in ADAR1 using an approach developed by Alan Herbert.[28][29]Crystallographic and NMR studies confirmed the biochemical findings that this domain bound Z-DNA in a non-sequence-specific manner.[30][31][32] Related domains were identified in a number of other proteins through sequence homology.[29] The identification of the Zα domain provided a tool for other crystallographic studies that lead to the characterization of Z-RNA and the B–Z junction. Biological studies suggested that the Z-DNA binding domain of ADAR1 may localize this enzyme that modifies the sequence of the newly formed RNA to sites of active transcription.[33][34] A role for Zα, Z-DNA and Z-RNA in defense of the genome against the invasion of Alu retro-elements in humans has evolved into a mechanism for the regulation of innate immune responses to dsRNA. Mutations in Zα are causal for human interferonopathies such as the Mendelian Aicardi-Goutières Syndrome.[35][18]

          Consequences of Z-DNA binding to vaccinia E3L protein[edit]

          As Z-DNA has been researched more thoroughly, it has been discovered that the structure of Z-DNA can bind to Z-DNA binding proteins through london dispersion and hydrogen bonding.[36] One example of a Z-DNA binding protein is the vaccinia E3L protein, which is a product of the E3L gene and mimics a mammalian protein that binds Z-DNA.[37][38] Not only does the E3L protein have affinity to Z-DNA, it has also been found to play a role in the level of severity of virulence in mice caused by vaccinia virus, a type of poxvirus. Two critical components to the E3L protein that determine virulence are the N-terminus and the C-terminus. The N-terminus is made of up a sequence similar to that of the Zα domain, also called Adenosine deaminase z-alpha domain, while the C-terminus is composed of a double stranded RNA binding motif.[37] Through research done by Kim, Y. et al. at the Massachusetts Institute of Technology, it was shown that replacing the N-terminus of the E3L protein with a Zα domain sequence, containing 14 Z-DNA binding residues similar to E3L, had little to no effect on pathogenicity of the virus in mice.[37] In Contrast, Kim, Y. et al. also found that deleting all 83 residues of the E3L N-terminus resulted in decreased virulence. This supports their claim that the N-terminus containing the Z-DNA binding residues is necessary for virulence.[37] Overall, these findings show that the similar Z-DNA binding residues within the N-terminus of the E3L protein and the Zα domain are the most important structural factors determining virulence caused by the vaccinia virus, while amino acid residues not involved in Z-DNA binding have little to no effect. A future implication of these findings includes reducing Z-DNA binding of E3L in vaccines containing the vaccinia virus so negative reactions to the virus can be minimized in humans.[37]

          Furthermore, Alexander Rich and Jin-Ah Kwon found that E3L acts as a transactivator for human IL-6, NF-AT, and p53 genes. Their results show that HeLa cells containing E3L had increased expression of human IL-6, NF-AT, and p53 genes and point mutations or deletions of certain Z-DNA binding amino acid residues decreased that expression.[36] Specifically, mutations in Tyr 48 and Pro 63 were found to reduce transactivation of the previously mentioned genes, as a result of loss of hydrogen bonding and london dispersion forces between E3L and the Z-DNA.[36] Overall, these results show that decreasing the bonds and interactions between Z-DNA and Z-DNA binding proteins decreases both virulence and gene expression, hence showing the importance of having bonds between Z-DNA and the E3L binding protein.

          Comparison geometries of some DNA forms[edit]

          Side view of A-, B-, and Z-DNA.
          The helix axis of A-, B-, and Z-DNA.
          A-form B-form Z-form
          Helix senseright-handedright-handedleft-handed
          Repeating unit1 bp1 bp2 bp
          Rotation/bp32.7°34.3°30°
          bp/turn111012
          Inclination of bp to axis+19°−1.2°−9°
          Rise/bp along axis2.3 Å (0.23 nm)3.32 Å (0.332 nm)3.8 Å (0.38 nm)
          Pitch/turn of helix28.2 Å (2.82 nm)33.2 Å (3.32 nm)45.6 Å (4.56 nm)
          Mean propeller twist+18°+16°
          Glycosyl angleantiantiC: anti,
          G: syn
          Sugar puckerC3′-endoC2′-endoC: C2′-endo,
          G: C3′-endo
          Diameter23 Å (2.3 nm)20 Å (2.0 nm)18 Å (1.8 nm)

          See also[edit]

          References[edit]

          1. ^Mitsui, Y.; Langridge, R.; Shortle, B. E.; Cantor, C. R.; Grant, R. C.; Kodama, M.; Wells, R. D. (1970). "Physical and enzymatic studies on poly d(I–C)·poly d(I–C), an unusual double-helical DNA". Nature. 228 (5277): 1166–1169. doi:10.1038/2281166a0. PMID 4321098. S2CID 4248932.
          2. ^Pohl, F. M.; Jovin, T. M. (1972). "Salt-induced co-operative conformational change of a synthetic DNA: equilibrium and kinetic studies with poly(dG-dC)". Journal of Molecular Biology. 67 (3): 375–396. doi:10.1016/0022-2836(72)90457-3. PMID 5045303.
          3. ^Thamann, T. J.; Lord, R. C.; Wang, A. H.; Rich, A. (1981). "High salt form of poly(dG–dC)·poly(dG–dC) is left handed Z-DNA: raman spectra of crystals and solutions". Nucleic Acids Research. 9 (20): 5443–5457. doi:10.1093/nar/9.20.5443. PMC 327531. PMID 7301594.
          4. ^Wang, A. H.; Quigley, G. J.; Kolpak, F. J.; Crawford, J. L.; van Boom, J. H.; van der Marel, G.; Rich, A. (1979). "Molecular structure of a left-handed double helical DNA fragment at atomic resolution". Nature. 282 (5740): 680–686. Bibcode:1979Natur.282..680W. doi:10.1038/282680a0. PMID 514347. S2CID 4337955.
          5. ^ abHa, S. C.; Lowenhaupt, K.; Rich, A.; Kim, Y. G.; Kim, K. K. (2005). "Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases". Nature. 437 (7062): 1183–1186. Bibcode:2005Natur.437.1183H. doi:10.1038/nature04088. PMID 16237447. S2CID 2539819.
          6. ^Placido, D.; Brown, B. A., II; Lowenhaupt, K.; Rich, A.; Athanasiadis, A. (2007). "A left-handed RNA double helix bound by the Zalpha domain of the RNA-editing enzyme ADAR1". Structure. 15 (4): 395–404. doi:10.1016/j.str.2007.03.001. PMC 2082211. PMID 17437712.
          7. ^Hall, K.; Cruz, P.; Tinoco, I., Jr; Jovin, T. M.; van de Sande, J. H. (Oct 1984). "'Z-RNA'—a left-handed RNA double helix". Nature. 311 (5986): 584–586. Bibcode:1984Natur.311..584H. doi:10.1038/311584a0. PMID 6482970. S2CID 4316862.
          8. ^de Rosa, M.; de Sanctis, D.; Rosario, A. L.; Archer, M.; Rich, A.; Athanasiadis, A.; Carrondo, M. A. (May 2010). "Crystal structure of a junction between two Z-DNA helices". Proceedings of the National Academy of Sciences. 107 (20): 9088–9092. Bibcode:2010PNAS..107.9088D. doi:10.1073/pnas.1003182107. PMC 2889044. PMID 20439751.
          9. ^Zhang, H.; Yu, H.; Ren, J.; Qu, X. (2006). "Reversible B/Z-DNA transition under the low salt condition and non-B-form poly(dA)poly(dT) selectivity by a cubane-like europium-L-aspartic acid complex". Biophysical Journal. 90 (9): 3203–3207. Bibcode:2006BpJ....90.3203Z. doi:10.1529/biophysj.105.078402. PMC 1432110. PMID 16473901.
          10. ^Ho, P. S.; Ellison, M. J.; Quigley, G. J.; Rich, A. (1986). "A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences". EMBO Journal. 5 (10): 2737–2744. doi:10.1002/j.1460-2075.1986.tb04558.x. PMC 1167176. PMID 3780676.
          11. ^ abChamp, P. C.; Maurice, S.; Vargason, J. M.; Camp, T.; Ho, P. S. (2004). "Distributions of Z-DNA and nuclear factor I in human chromosome 22: a model for coupled transcriptional regulation". Nucleic Acids Research. 32 (22): 6501–6510. doi:10.1093/nar/gkh988. PMC 545456. PMID 15598822.
          12. ^Wang, Andrew H.-J.; Quigley, Gary J.; Kolpak, Francis J.; Crawford, James L.; van Boom, Jacques H.; van der Marel, Gijs; Rich, Alexander (December 1979). "Molecular structure of a left-handed double helical DNA fragment at atomic resolution". Nature. 282 (5740): 680–686. Bibcode:1979Natur.282..680W. doi:10.1038/282680a0. ISSN 0028-0836. PMID 514347. S2CID 4337955.
          13. ^ abcLee, Juyong; Kim, Yang-Gyun; Kim, Kyeong Kyu; Seok, Chaok (2010-08-05). "Transition between B-DNA and Z-DNA: Free Energy Landscape for the B−Z Junction Propagation". The Journal of Physical Chemistry B. 114 (30): 9872–9881. CiteSeerX 10.1.1.610.1717. doi:10.1021/jp103419t. ISSN 1520-6106. PMID 20666528.
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          Источник: https://en.wikipedia.org/wiki/Z-DNA

          Abstract

          The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore-forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Examination of additional point mutants unexpectedly revealed that the CTS of Bim directly interacts with Bax, is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.

          Introduction

          Apoptosis is a highly conserved form of programmed cell death that can be triggered by extrinsic or intrinsic signals. It plays a fundamental role in maintaining homeostasis by eliminating old, excessive or dysfunctional cells in multicellular organisms (Kerr et al., 1972). Defective regulation of apoptosis has been found in many diseases (Favaloro et al., 2012) and is considered one of the hallmarks of cancer (Hanahan and Weinberg, 2011).

          Bcl-2 family proteins play a decisive role in apoptosis initiated by intrinsic signaling by regulating the integrity of the mitochondrial outer membrane (MOM). Commitment to apoptosis is generally regarded as due to MOM permeabilization (MOMP) releasing cytochrome c and pro-apoptotic factors from the intermembrane space into the cytoplasm. These factors activate the executioner caspases that mediate cell death (Chipuk et al., 2006). Direct interactions between Bcl-2 family proteins govern both initiation and inhibition of MOMP (Kale and Osterlund, 2017). The Bcl-2 family of proteins that regulate apoptosis includes the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1 that inhibit the process and share four Bcl-2 homology domains. These homology domains, referred to as BH domains, are also shared by the pro-apoptotic proteins Bax and Bak that permeabilize the MOM directly. Both pro- and anti-apoptotic multi-domain Bcl-2 family proteins are regulated by direct binding interactions with a group of proteins including Bim, Bid, Puma, Hrk, Bad and Noxa that contain a single region of homology, the Bcl-2 homology domain number 3, and are therefore referred to collectively as BH3-proteins. These proteins promote apoptosis by releasing sequestered activated Bax, Bak and BH3-proteins that activate Bax and Bak from one or more of the anti-apoptotic proteins. The subset of BH3 proteins that bind to and activate Bax or Bak include Bid, Bim and Puma (Chi et al., 2014). Thus far, the biochemical basis for the differences between BH3-proteins that inhibit anti-apoptotic proteins and those that activate Bax and Bak has been attributed entirely to differences in affinities of the BH3-domain for the BH3-peptide binding sites on multi-domain pro- and anti-apoptotic proteins. However, static affinities and variations in expression levels permit only coarse regulation of cell death. Changes in the equilibrium binding of Bcl-2 family proteins on the MOM enable finer control. For example, at physiologic concentrations, the BH3-protein Bid only activates Bax after Bid has bound to a membrane and undergone a specific conformational change (Lovell et al., 2008; Shamas-Din et al., 2013a). Binding to membranes also enables interaction of Bid with MTCH2 on the MOM to greatly accelerate the Bid conformational change that results in Bax activation (Shamas-Din et al., 2013a). However, it remains unclear whether membrane interactions by other BH3-proteins like Bim contribute to Bax activation.

          The BH3-protein Bim is an important mediator of apoptosis initiated by many intracellular stressors (Concannon et al., 2010; Mahajan et al., 2014; Puthalakath et al., 2007). Three major isoforms of Bim result from alternative mRNA splicing: BimEL, BimL, and BimS (O'Connor et al., 1998). All three isoforms include the BH3-domain required for binding other Bcl-2 family proteins, and a C-terminal sequence (CTS) that binds the protein to the MOM (Wilfling et al., 2012). BimEL and BimL also share a dynein light chain binding motif (LC1) that sequesters these isoforms at the cytoskeleton (Lei and Davis, 2003). However, recent evidence also suggests that the LC1 sequence mediates Bim oligomerization by binding to the DLC1 protein (Singh et al., 2017). Isoform BimS does not contain the LC1-binding motif (Lei and Davis, 2003), and is rarely present in healthy cells, while BimL and BimEL are present in most tissue types (O'Reilly et al., 2000). Bim has a particularly important function as a regulator of anti-apoptotic proteins, as it binds and thereby inhibits by mutual sequestration all known anti-apoptotic proteins (Chen et al., 2005; Shamas-Din et al., 2013b). Until recently, it was unknown why Bim binds to Bcl-XL with sufficient affinity to resist displacement by small molecule BH3-mimetics, while other BH3-proteins, such as Bad, are displaced (Aranovich et al., 2012). In addition to interactions via the BH3-domain, residues within the Bim CTS bind to Bcl-XL, and thereby increase the affinity of the interaction by ‘double-bolt locking’ providing an explanation for the observations with BH3 mimetic drugs (Liu et al., 2019). Here, we investigated whether the CTS of Bim also contributes to the functional and physical interactions between Bim and Bax.

          We demonstrate that both primary cells and cell lines have a range of apoptotic responses to the expression of a truncated BimL protein lacking the CTS (BimL-dCTS), while expression of full-length BimL was sufficient to kill all of these cells. To determine the molecular mechanism that underlies this difference, the two pro-apoptotic functions of Bim; activation of Bax and inhibition of Bcl-XL, were quantified using purified full-length BimL and BimL mutant proteins and cell-free assays. Replacing the CTS of Bim with an alternative tail-anchor that binds the protein to mitochondrial membranes did not fully restore Bax activation function, demonstrating that sequences within the Bim CTS rather than membrane binding contribute to Bax activation. Site-directed mutagenesis of the Bim CTS also revealed residues important for binding to membranes that were not required for Bax activation (e.g. I125). Furthermore, specific residues within the CTS were identified that are required for BimL to efficiently activate Bax, but that are not required for BimL to bind to and inhibit Bcl-XL. Evidence in cell free assays demonstrated that BimL CTS residues L129 and I132 physically interact with Bax in the BH3 binding groove and are required for Bim to activate Bax. Together, our data demonstrates that the unusual sequence of the CTS of Bim separately controls both membrane binding and Bax activation.

          Results

          The CTS of Bim variably contributes to the pro-apoptotic activity of Bim in different cell lines

          Removing the CTS from Bim abrogates pro-apoptotic activity in HEK293 cells (Weber et al., 2007). While this observation has generally been ascribed to loss of binding of Bim to MOM our observation that the CTS is also involved in binding BimEL to Bcl-XL (Liu et al., 2019) suggested that there may be other explanations for the loss of pro-apoptotic activity for Bim when the CTS is removed. To determine the contribution of the Bim CTS to pro-apoptotic activity, a BimL mutant was generated in which the previously characterized membrane binding domain (carboxyl-terminal residues P121- H140) were deleted (BimL-dCTS) (Wilfling et al., 2012; Liu et al., 2019). This mutant was expressed in cells and the effectiveness of induction of cell death was compared to expression of full-length BimL by confocal microscopy. To detect expression of the constructs in live cells, they included an N-terminally fused Venus fluorescent protein (indicated by a superscripted v in the name). Thus, a construct in which Venus was fused to the amino-terminus of BimL is referred to here as VBimL while the mutant lacking the CTS is VBimL-dCTS. As an inactive control, we used VBimL-4E a mutant in which four conserved hydrophobic residues in the BH3-domain of BimL were replaced with glutamate, thereby preventing binding to all other multi-BH domain Bcl-2 family proteins (Chen et al., 2005; Liu et al., 2019).

          To assay pro-apoptotic activity, the constructs were expressed in primary cells and cell lines and both expression and cell death were measured using confocal microscopy. Apoptosis was assessed by detecting externalization of phosphatidylserine by Annexin V staining in cells expressing detectable levels of VBimL or the VBimL mutants as measured by Venus fluorescence. As a positive control for activation of Bax VtBid, the activated form of the BH3-protein Bid fused to the C-terminus of the Venus fluorescent protein was also expressed in cells. As expected, expression of VBimL induced apoptosis in all cell types tested, while the negative control protein VBimL-4E did not (Figure 1A). Similarly, VtBid induced apoptosis in all the cell types except HEK293 cells. As reported previously for Bim-dCTS, the fluorescent version (VBimL-dCTS) failed to induce cell death in HEK293 cells (Weber et al., 2007). In contrast, expression of VBimL-dCTS induced apoptosis to levels similar to VBimL in HCT116, BMK and MEF cells but may have reduced potency in CAMA-1 cells. Comparing the AnnexinV intensities for individual cells at a variety of equivalent expression levels of the Bim mutants across the different cell types revealed that the CTS of Bim was required for the pro-apoptotic activity of Bim in HEK293 cells (Figure 1—figure supplement 1).

          To determine if this difference in response to VBimL-dCTS expression is a function of the extent to which the apoptotic machinery is loaded in MOM, mitochondria were purified from cells resistant (HEK293) and sensitive (MEF) to VBimL-dCTS expression and assayed by BH3-profiling (Potter and Letai, 2016). This assay measures loading of anti-apoptotic proteins with BH3-proteins or active Bax/Bak. Unlike BH3-profiling experiments conducted with BH3-peptides, in these experiments purified full-length proteins were used. Thus, purified cBid, BimL, BimL-dCTS, Bad and Noxa proteins were incubated with mitochondria from each of the cell lines and MOMP was measured by separating supernatant and pellet fractions for each reaction, and immunoblotting for cytochrome c released from the intermembrane space as previously described (Pogmore et al., 2016). Immunoblots were quantified and MOMP assessed as % cytochrome c released (Figure 1B). As expected from the data in Figure 1A, addition of recombinant BimL was sufficient to induce cytochrome c release from mitochondria from both HEK293 and MEF cells. However, addition of BimL-dCTS induced cytochrome c release only in the MEF mitochondria confirming that resistance to BimL-dCTS in HEK293 cells is manifest at mitochondria. Mitochondria purified from HEK293 cells were more sensitive to BimL protein than to recombinant cBid, a phenomenon that is also seen when VtBid is expressed in these cells (Figure 1A). This result may be due to the inherent differences between Bim and Bid reported previously (Sarosiek et al., 2013). Nevertheless, in HEK293 cells, VtBid was more active than BimL-dCTS and functionally equivalent to VBimL in every other cell line tested.

          One potential explanation for the difference in response to BimL-dCTS and BimL is that the mitochondria in the cell lines have different dependencies on multi-domain anti-apoptotic proteins for survival, a phenomenon known as priming. If BimL-dCTS has lost one of the functions of Bim such as activating Bax or Bak or inhibiting one of the multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 it would be expected to have different activities on mitochondria with different priming. Therefore, to better understand why BimL-dCTS can only permeabilize MEF mitochondria and not mitochondria from HEK293 cells, we compared the sensitivity of mitochondria from the two cell types to addition of BH3-proteins Bad or Noxa that inhibits Bcl-2 and Bcl-XL or Mcl-1, respectively, but that do not activate Bax or Bak (Kale and Osterlund, 2017). Incubation of full-length Bad and/or Noxa with mitochondria from HEK293 cells failed to induce cytochrome c release, while the addition of Noxa or Bad was sufficient to permeabilize MEF mitochondria (Figure 1B). This data suggests that HEK293 cells not depend on expression of Bcl-2, Bcl-XL or Mcl-1 sequestering active Bax, Bak or their BH3-activators while mitochondria from MEFs depend on expression of Mcl-1 and Bcl-XL to prevent apoptosis (Lessene et al., 2013). The results further suggest that removal of the CTS from BimL results in a mutant protein that only kills cells dependent on one or more multi-domain anti-apoptotic proteins for survival. That BimL-dCTS does not kill HEK293 cells further suggests that it does not activate sufficient Bax or Bak to overcome the unoccupied anti-apoptotic proteins in this cell line. In this way, BimL-dCTS functions as a sensitizer similar to proteins like Bad and Noxa. However, unlike other relatively specific sensitizer proteins, the BH3-region of BimL-dCTS binds to and inhibits Bcl-2, Bcl-XL and Mcl-1. Indeed, we have shown that in live cells BimEL-dCTS binds to and inhibits Bcl-2 and Bcl-XL but is more easily displaced than BimEL by small molecule BH3 mimetics (Liu et al., 2019).

          Full-length BimL is required to kill cultures of primary cortical neurons

          Our data with cell lines and their respective purified mitochondria suggests that BimL-dCTS does not kill cells that do not depend on anti-apoptotic proteins for survival. To test this in a more biologically relevant system, we cultured primary murine cortical neurons and assayed their response to expression of the BimL mutants. To enable regulated expression in primary cortical neurons the coding regions for VBimL, VBimL-4E, and VBimL-dCTS were cloned into a tetracycline-responsive lentiviral vector, and introduced into primary cortical neuron cultures through lentiviral infection. After culture for 16 days in vitro, BimL expression was induced in the neurons by the addition of doxycycline. Neuronal cell death was assayed using confocal microscopy after staining neurons with TMRE, a dye that only accumulates in active mitochondria. Thus, a lack of TMRE dye accumulation (TMRE negative) indicates loss of mitochondrial transmembrane potential and in response to expression of a BH3-protein is an early indication of commitment to cell death. Quantification of Venus-expressing neuronal cell bodies revealed that as expected VtBid and VBimL expression killed cultured primary neurons while VBimL-4E did not (Figure 2A–B). However, the expression of VBimL-dCTS was largely ineffective to induce cell death in cultured primary cortical neurons (Figure 2B). Our data is consistent with previous reports suggesting that primary murine cultures of hippocampal neurons become resistant to induction of apoptosis by external stimuli over time in culture. This resistance has been reported to be due to a difference in Bcl-2 family protein expression that results in decreased mitochondrial ‘priming’, explaining why our cultures of primary cortical neurons are resistant to VBimL-dCTS (Sarosiek et al., 2017).

          To determine if resistance to induction of cell death by BimL-dCTS is due to differential sensitivity of neuronal mitochondria to induction of MOMP by BimL and BimL-dCTS, mitochondria were isolated from embryonic day 15 (E15) mouse brains, the same age used to culture primary cortical neurons. Brain mitochondria were used instead of isolating mitochondria from neuronal cultures due to the low yield from primary cultured neurons. Untreated mitochondria from day E15 brain released only low levels of cytochrome c. As expected, addition of 0.1 nM recombinant BimL was sufficient to elicit MOMP as measured by cytochrome c release and detection in the supernatant. In contrast, 100 times more BimL-dCTS (10 nM) failed to induce MOMP (Figure 2C).

          Taken together our data suggest that BimL-dCTS kills cells in which the mitochondria are sensitive to inhibition of anti-apoptotic proteins by sensitizers such as Bad and Noxa. Thus, BimL-dCTS did not permeabilize mitochondria extracted from HEK293 cells or E15 whole murine brains, and as a result, BimL-dCTS expression did not kill HEK293 cells or primary cultures of cortical neurons. This finding suggests that inhibition of anti-apoptotic proteins is not sufficient to kill these cells. Therefore, BimL-dCTS differs mechanistically from BimL as the latter kills both cell types resistant and sensitive to BimL-dCTS. Compared to BimL, BimL-dCTS is missing the membrane-binding domain and therefore is not expected to localize at mitochondria (Liu et al., 2019); however, the relationship between Bim binding to membranes and Bim-mediated Bax activation has not been extensively studied. To determine how the molecular mechanism of BimL-dCTS differs from BimL the activities of the proteins were analyzed using cell-free assays.

          The Bim CTS mediates BimL binding to both Bax and membranes

          To investigate the pro-apoptotic mechanism of BimL and BimL-dCTS without interference from other cellular components, both were purified as full-length recombinant proteins and assayed using liposomes and/or isolated mitochondria. To measure direct-activation of Bax by Bim, either BimL or BimL-dCTS was incubated with recombinant Bax and liposomes encapsulating the dye and quencher pair: ANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt) and DPX (p-Xylene-Bis-Pyridinium Bromide). In this well-established assay (Chi et al., 2014), increasing amounts of BimL activated Bax results in membrane permeabilization measured as an increase in fluorescence due to the release and separation of encapsulated dye and quencher (Figure 3A). This result is consistent with previous observations that picomolar concentrations of BimL induce Bax-mediated membrane permeabilization (Sarosiek et al., 2013). In contrast, three orders of magnitude higher concentrations of BimL-dCTS were required to induce Bax-mediated liposome permeabilization (Figure 3A), suggesting that either or both of binding to membranes and the specific CTS of Bim are required for efficient Bax activation. As expected, similar results were obtained for Bax-mediated release of mitochondrial intermembrane space proteins (Figure 3B). For these experiments, MOMP was measured as release of the fluorescent protein mCherry fused to the N-terminal mitochondrial import signal of SMAC (SMAC-mCherry) from the intermembrane space of mitochondria (Shamas-Din et al., 2014). Similar to the results with liposomes (Figure 3A), and mitochondria from cell lines (Figures 1–2) BimL but not BimL-dCTS triggered Bax mediated SMAC-mCherry release from mitochondria isolated from Bax - /- Bak-/-cells (Figure 3B). In experiments with liposomes and mitochondria, very small amounts of Bim were sufficient to trigger membrane permeabilization because once activated, Bax recruits and activates additional Bax molecules (Tan et al., 2006). To assess the impact of the Bim CTS on the interaction between Bim and Bax, binding was measured using Förster resonance energy transfer (FRET). For these experiments, recombinant BimL proteins were labeled with the donor fluorophore Alexa568, while Bax was labeled with the acceptor fluorophore Alexa647. Unexpectedly, and unlike the BH3-only protein tBid (Lovell et al., 2008), BimL bound to Bax even in the absence of membranes (Figure 3C), while BimL-dCTS had no relevant Bax binding in the presence or absence of mitochondrial-like liposomes (Figure 3C–D). Binding of Bim to Bax in solution suggests that the CTS of Bim may be directly involved in Bim-Bax heterodimerization independent of Bim binding to membranes.

          To confirm in our system that the labeled BimL proteins bind to membranes via the CTS sequence, binding of Alexa568-labeled recombinant BimL and BimL-dCTS to DiD labeled liposomes was measured by FRET (Figure 4A). In these experiments, DiD serves as an acceptor for energy transfer from Alexa568-labeled BimL. The same approach was used to quantify BimL binding to mitochondrial outer membranes with mitochondria isolated from BAK-/-mouse liver (Figure 4B), which lack Bax and Bak (Shamas-Din et al., 2013a). In both cases, BimL spontaneously bound to membranes with picomolar affinity, while stable binding of BimL-dCTS to liposomes and mitochondria was not-detectable (Figure 4A–B). Furthermore, BimL-dCTS again had no relevant binding to Bax even in the presence of purified mitochondria (Figure 4C).

          Taken together, our data strongly suggest that the CTS of Bim is required for both BimL to bind to membranes in vitro and for binding Bax with or without membranes. Alternatively purified BimL-dCTS may be completely non-functional. To demonstrate that purified BimL-dCTS binds to and inhibits Bcl-XL as shown for VBimL-dCTS expressed in cells (Figure 1) and in Liu et al. (2019), inhibition of Bcl-XL was measured using liposomes and mitochondria.

          The CTS is not required for BimL to inhibit Bcl-XL

          In addition to direct Bax activation, Bim promotes apoptosis by binding to Bcl-XL and displacing either activator BH3-proteins (Mode 1) or activated Bax or Bak (Mode 2) (Llambi et al., 2011). In the ANTS/DPX liposome dye release assay, BimL-dCTS was functionally comparable to the well-established Bcl-XL inhibitory BH3-protein Bad in reversing Bcl-XL-mediated inhibition of cBid (Figure 5A) or Bax (Figure 5B). Consistent with the observation that BimL-dCTS was less resistant to displacement by BH3 mimetics in live cells, in cell-free assays BimL-dCTS was also less effective than BimL at displacing cBid or Bax from Bcl-XL (Liu et al., 2019). Nevertheless, when assayed with mitochondria BimL-dCTS disrupted the interaction between tBid and Bcl-XL resulting in Bax activation and permeabilization of mitochondria as measured by cytochrome c release (Figure 5C, solid black line). This activity is due to inhibition of Bcl-XL function, as in controls without Bcl-XL the same concentration of BimL-dCTS did not directly activate sufficient Bax to mediate MOMP (Figure 5C, dashed black line). Thus, purified BimL-dCTS is functional and can initiate MOMP by displacing direct-activators (Mode 1) or activated Bax (Mode 2) from Bcl-XL (Figure 5A–C). Finally, BimL-dCTS labeled with Alexa568 retained high-affinity binding for Bcl-XL labeled with Alexa647 both in solution (Kd <16 nM) and on membranes (~35 nM apparent Kd on liposomes and on mitochondria) as measured by FRET (Figure 6B).

          Different residues in the Bim CTS regulate membrane binding and Bax activation

          To identify which residues in the Bim CTS mediate binding to membranes and/or Bax we generated a series of point mutations. Sequence analysis using HeliQuest software (Gautier et al., 2008) predicts that the Bim CTS forms an amphipathic α-helix (Figure 6A). Two arginine residues (R130 and 134) are predicted to be on the same hydrophilic side of the helix, whereas hydrophobic residues (e.g. I125, L129, I132) face the other side (Figure 6A). To determine the functional importance of these residues, Bim CTS mutants were created including: BimL-CTS2A in which R130 and R134 were mutated to alanine; and a series of single hydrophobic residue substitutions by glutamate (V124E, I125E, L129E, and I132E) (Figure 6A). To compare the effects of the CTS mutations on BimL-binding interactions and function, we measured by FRET the Kds for the various binding interactions and the activities of the mutants to promote Bax-mediated liposome permeabilization as EC50’s for ANTS release (Figure 6B and Figure 6—figure supplement 1A–E).

          Mutation of individual hydrophobic residues on the hydrophobic side of the Bim CTS (BimL-I125E, BimL-L129E or BimL-I132E) abolished binding to membranes (Figure 6B). In contrast, mutations on the other side of the helix including BimL-V124E and BimL-CTS2A had less effect on Bim binding to membranes (Figure 6B). Despite the dramatic changes in affinity for membranes among Bim CTS mutants, the mutations did not abolish binding to Bax both in the presence and absence of membranes (Figure 6B). Indeed most of the mutants had Kd values for binding to Bax of less than 100 nM and to our surprise many of them bound to Bax better in solution compared to when membranes were present. The presented Kds under ‘membranes present’ is an estimation of the effective Kd (combined solution and two-dimensional Kds), as we are not able to precisely determine the quantity of protein-complexes that form in solution or on the membrane. Nevertheless, this data reflects the situation in cells and further confirms that binding to membranes and Bax are independent functions of the Bim CTS . In the case of BimL-I125E, a mutant that activates Bax to permeabilize liposomes, the initial interaction with Bax must occur in solution as neither protein spontaneously binds to membranes (Figure 6B).

          Unexpectedly, there was not a good correlation between BimL binding to membranes and Bax activation. For example, while BimL bound to membranes with a Kd of 31 pM, BimL-CTS2A and BimL-I125E bound to membranes with Kds of ~600 and>1000 pM, respectively yet both mutants triggered Bax-mediated membrane permeabilization, demonstrating that specific residues in the CTS rather than binding to membranes enabled BimL to mediate Bax activation. Moreover, BimL binding to Bax was also not sufficient to activate Bax efficiently. BimL-L129E and BimL-I132E are two Bim mutants that do not bind membranes, retain reasonable affinities for Bax in the presence of membranes (Kds ~ 100–200 nM), but were unable to activate Bax (Figure 6B). These results indicate that these two residues play a key function in Bax activation. As expected, the negative control BimL-4E mutant does not bind to nor activate Bax even though its CTS is intact and the protein binds membranes (Figure 6B). This result confirms the essential role of the BH3-domain and suggests that the Bim CTS provides a secondary role in Bax binding rather than providing an independent high affinity binding site that is sufficient to activate Bax.

          Both functional and binding assays for the various point mutants suggest that specific residues in the Bim CTS participate in Bim-Bax protein interactions that lead to Bax activation; however, these mutants did not clearly separate the membrane binding function of the CTS of Bim from a potential function in Bax activation. Thus, it remains possible that restoring membrane binding to BimL-dCTS would be sufficient to restore Bax activation function. To address this, we fused the mitochondrial tail-anchor from mono-amine oxidase (MAO residues 490–527, UniProt: P21397-1) to the C-terminus of BimL-dCTS to restore membrane binding with a sequence unlikely to contribute to Bax activation directly. This protein, BimL-dCTS-MAO, and BimL bound to mitochondrial-like liposomes and purified mitochondria (Figure 7A and Figure 7—figure supplement 1A respectively). As expected, a population of these recombinant Bim proteins remains in solution. To directly assess the Bim CTS contribution to the binding and activation of Bax on the membrane surface, we incubated a defined amount of recombinant Alexa568-labeled BimL or BimL-dCTS-MAO proteins with 0.5 mg/mL ANTS/DPX filled liposomes, then isolated the liposomes using size-exclusion chromatography. Using this procedure, we excluded all recombinant Bim that remained in solution, and obtained the membrane-bound BimL or BimL-dCTS-MAO at a concentration of ~5 nM as calculated based on Alexa568 fluorescence intensity (Precise concentrations labeled in Figure 7B). This equates to 0.6 Bim molecules per liposome. Addition of Bax to these liposomes resulted in ANTS/DPX release for roughly 60% of the liposomes in incubations containing BimL-membrane-bound liposomes, but no significant release from the liposomes in incubations containing BimL-dCTS-MAO-bound liposomes (Figure 7B), suggesting that the Bim CTS contributes to the activation of Bax even on the membrane surface.

          To directly measure binding to Bax, Alexa568-labeled BimL and BimL-dCTS-MAO were incubated with increasing concentrations of Alexa647-labeled Bax protein. In the absence of membranes, no detectable FRET was measured between Alexa568-labeled BimL-dCTS-MAO with Alexa647-labeled Bax (Figure 7C), suggesting that the specific sequence of the Bim CTS contributes to the Bim-Bax interaction in solution. However, when Alexa568-labeled BimL-dCTS-MAO bound to the liposome membrane was isolated by size-exclusion chromatography as described above, a FRET signal was detected between it and Alexa647-labeled Bax. We also detected FRET between Alexa568-labeled BimL-dCTS-MAO and Alexa647-labeled Bax in the presence of purified mitochondria (Figure 7—figure supplement 1B). Although these incubations contained both soluble and membrane-bound Bim protein, we conclude the interaction occurred on the MOM as no FRET signal was detected between these proteins in solution (Figure 7C). Despite measureable binding between BimL-dCTS-MAO and Bax on the liposome membrane (Figure 7D), BimL retained a higher affinity than BimL-dCTS-MAO for Bax (Kds of 21 nM and 49.6 nM, respectively). Together these data suggest that the CTS of Bim contributes to both binding to (Figure 7D) and activation of Bax (Figure 7B) on the liposome membrane.

          Residues within the Bim CTS are proximal to Bax in solution and on mitochondrial membranes

          Our binding and mutagenesis data suggest that the Bim CTS binds to and activates Bax in solution and on membranes. To detect this binding interaction, we used a photocrosslinking approach, in which a BimL protein was synthesized with a photoreactive probe attached to a single lysine residue positioned in the CTS using an in vitro translation system containing 5-azido-2-nitrobenzoyl-labled Lys-tRNALys that incorporates the lysine analog (εANB-Lys) into the polypeptide when a lysine codon in the BimL mRNA is encountered by the ribosome. The BimL synthesized in vitro was also labeled by 35S via methionine residues enabling detection of BimL monomers and photoadducts by phosphor-imaging.

          The radioactive, photoreactive BimL protein was incubated with a recombinant His6-tagged Bax protein in the presence of mitochondria isolated from BAK-/- mouse liver lacking endogenous Bax and Bak to prevent competition and increase BimL-Bax protein interactions. Mitochondrial proteins were then separated from the soluble ones by centrifugation. Both soluble and mitochondrial fractions were photolyzed to activate the ANB probe generating a nitrene that can react with atoms in close proximity (<12 Å from the Cα of the lysine residue). Thus, for photoadducts to form, the atoms of the bound Bax molecule are likely to be located in or near the binding site for the Bim CTS. The resulting photoadduct between the BimL and the His6-tagged Bax was enriched by Ni2+-chelating agarose resin and separated from the unreacted BimL and Bax monomers using SDS-PAGE. The 35S-labeled BimL in the photoadduct with His6-tagged Bax and BimL monomer bound to the Ni2+-beads specifically via the His6-tagged Bax or nonspecifically were detected by phosphor-imaging. A BimL-Bax-specific photoadduct was detected when the ANB probe was located at four different positions in the Bim CTS on both hydrophobic and hydrophilic surfaces of the potential α-helix (Figure 8A). These photoadducts have the expected molecular weight for the Bim-Bax dimer, and were not detected or greatly reduced when the ANB probe, the light, or the His6-tagged Bax protein was omitted (Figure 8A). Consistent with the BimL-Bax interaction detected by FRET in both solution and membranes, the BimL-Bax photocrosslinking occurred in both soluble and mitochondrial fractions. Less photocrosslinking occurred in the mitochondrial fraction likely due to the fact that in membranes homo-oligomerization of activated Bax competes with hetero-dimerization between BimL and Bax.

          As expected, when the ANB probe was positioned in the Bim-BH3 domain as a positive control BimL-Bax photocrosslinking was detected in both soluble and mitochondrial fractions (Figure 8B). Crosslinking with the Bim BH3-domain is consistent with the BH3 interaction with the canonical groove or trigger pocket that is well supported by experimental evidence including co-crystal structures and NMR models (Gavathiotis et al., 2008; Robin et al., 2015). Furthermore, loss of photocrosslinking for BimL mutants with the BH3-4E mutation that abolished binding to Bax demonstrates that direct binding between the proteins is required for crosslinking to be detectable (Figure 8C). Therefore, the crosslinking data suggests that similar to the BH3-domain, the Bim CTS binds to Bax. To further demonstrate that the CTS of Bim binds to Bax independent of both membrane binding and Bax activation, the experiment was repeated with BimL-L129E, a mutant that binds Bax without activating it and that does not bind membranes (Figure 6B). As shown in Figure 8C, the L129E mutant photocrosslinked to Bax in both the soluble and mitochondrial fractions. Furthermore, this mutant also photocrosslinked to Bcl-XL (Figure 8C), consistent with data demonstrating that the Bim CTS also binds to this anti-apoptotic protein (Figure 6B and Liu et al., 2019). The qualitative photocrosslinking data (Figure 8) and the quantitative FRET data (Figure 6B) obtained from the BimL proteins with and without the CTS or BH3-domain mutation are consistent, and both support a model in which the CTS interacts with membranes and binds to Bax, thereby enhancing BH3-domain mediated Bax activation.

          The Bim CTS binds to the BH3-binding pocket on Bax

          To identify the binding site for the Bim CTS in Bax, we used a chemical crosslinking approach. Unlike the photocrosslinking approach that does not reveal the location of the binding site, the chemical crosslinker bismaleimidohexane (BMH) contains two sulfhydryl reactive moieties separated by a 13 Å spacer, and thus formation of a BMH-crosslinked Bim-Bax dimer requires a cysteine in Bim that is in close proximity with another cysteine in the interacting Bax. Therefore, a successful crosslink indicates a close proximity between the two cysteines, potentially revealing the Bim-binding site in Bax.

          We used a structurally well-defined Bim-Bax interaction to validate this crosslinking approach. According to the crystal structure (PDB ID 4ZIE; Robin et al., 2015), the BH3-domain of Bim binds to the canonical groove of Bax. Our FRET data shows that BimL and Bax bind in solution and the binding is abolished by the 4E mutation in the Bim BH3-domain that eliminates the nonpolar interactions with the Bax groove. It is therefore expected that this Bim-Bax interaction is mediated by the BH3-domain and the groove, and according to the structure, a BMH molecule would be able to link a cysteine replacing Phe101 in the BH3-domain of Bim to a cysteine replacing Trp107 in the canonical groove of Bax. We thus synthesized the [35S]Met-labeled single-Cys Bim F101C and Bax W107C proteins in vitro, let them interact in solution, and subjected the sample to BMH crosslinking. When the products were analyzed by SDS-PAGE and phosphor-imaging two BMH-crosslinked products with molecular weights close to that of a BimL-Bax heterodimer were detected (Figure 9A, lane 4, indicated by open and closed triangles). The lower molecular weight band indicated by a closed triangle is the BMH-linked BimL-Bax heterodimer since it was absent in the control reactions when either the single-cysteine BimL or Bax was replaced by the respective cysteine-null (C0) protein (Figure 9A, lane 6 or 2). The higher molecular weight band indicated by an open triangle is the BMH-linked Bax homodimer since it was also present in the control reaction containing the single-cysteine Bax and the cysteine-null BimL (Figure 9A, lane 6). These results demonstrate the BMH crosslinking approach can detect the interaction of the BH3-domain of BimL with the canonical groove of Bax, and hence in principle it can be used to reveal the Bim CTS-binding site in Bax.

          Sequence analysis predicts that similar to the BH3-domain the Bim CTS forms an amphipathic α-helix (Figure 6A). Sequence alignment revealed a high similarity between the CTS and the BH3-domain as both have the same hydrophobic residues at the h0, h1, h2 and h3 positions, and the same polar or charged residue at the h1+two or h2+one position (Figure 9D). The Bim BH3 residues at these positions make critical contacts with the Bax canonical groove that are important for Bax activation (Robin et al., 2015; Weber et al., 2007). To determine whether the Bim CTS binds to the same Bax groove as the Bim BH3-domain, we performed BMH crosslinking using a Bim W137C mutant that has a single cysteine near the C-terminus of the CTS. Like the Bim F101C mutant with the cysteine near the C-terminus of the BH3-domain, Bim W137C crosslinked to Bax W107C (Figure 9A, lane 10, indicated by a closed triangle), suggesting that the BH3-binding groove is also a binding site for the Bim CTS. Consistent with this interpretation, this BimL-Bax heterodimer specific crosslinking did not occur in the control reaction with either single-Cys protein substituted by the respective cysteine-null protein (Figure 9A, lane 8 or 12), unlike the Bax homodimer specific crosslinking that also occurred in the control reaction with the single-cysteine Bax and cysteine-null BimL (Figure 9A, lane 6). Reciprocal immunoprecipitation by BimL and Bax specific antibodies further identified the crosslinked BimL-Bax heterodimer from the cysteine in the Bim CTS or BH3-domain to the cysteine in the Bax groove (Figure 9—figure supplement 1).

          To further define this noncanonical CTS-groove interaction, we repeated the crosslinking using other cysteine positions in the Bim CTS and additional cysteine mutants in the canonical groove in Bax. The only strong heterodimer specific crosslinking detected was between Bim M123C (a cysteine near the N-terminus of the CTS) and Bax W107C (Figure 9A, lane 14, indicated by a closed triangle). Since this Bax mutant was also crosslinked to the C-terminus of CTS via W137C, binding of the CTS to the groove seems to occur in either orientation. Additional weak but specific BimL to Bax crosslinking was detected from M123C to E69C and W137C to D98C providing further support for this flexible interaction (Figure 9—figure supplement 1).

          To determine whether the physical interaction between the Bim CTS or BH3-domain and the Bax groove detected by the crosslinking is functional, we tested the effect of the L129E mutation in the Bim CTS or the 4E mutation in the Bim BH3-domain on the crosslinking because both mutations greatly inhibited the activation of Bax by BimL (Figure 6B). We found that the L129E mutation reduced the Bim CTS to Bax interaction with the binding groove detected by the Bim W137C or W123C to Bax W107C crosslinking (compare the closed triangle-indicated band in Figure 9B, lane 10 or 14 with that in Figure 9A, lane 10 or 14). Since we used Bim F101C to detect the BH3-domain interaction with the Bax groove, and the F101 was changed to E in the 4E mutant, we generated Bim F101C/3E mutant to assess the effect of the BH3 mutation. As expected, the 3E mutation abolished Bim F101C crosslinking to Bax W107C, and hence, the BH3-interaction with the groove (Figure 9C, the closed triangle indicates the band in lane two that disappeared in lane 4 when the 3E mutant was used). Surprisingly, mutation of the CTS also inhibited the BH3-interaction with the groove (Figure 9B, lane four vs. Figure 9A, lane 4), while the BH3 mutation also abolished the CTS interaction with the groove (Figure 9C, lane 12 vs. lane 10), suggesting that the two interactions are not independent. The BH3-domain may contribute more than the CTS to the overall protein-protein interaction based on the severity of the effect of mutations on the crosslinking and FRET (Figure 6B). Together, the crosslinking data from these loss-of-function Bim mutants demonstrate that the CTS and BH3-interactions with the groove detected between the soluble Bim and Bax proteins are functionally important for Bim mediated activation of Bax.

          Bim CTS mutants that cannot activate Bax in vitro do not kill HEK293 cells

          Together, our data suggests that specific residues within the Bim CTS are involved in different aspects of BimL function. Residue I125 is required for Bim to bind to mitochondria but is of lesser importance in activating Bax. In contrast, residues L129 and I132 are not required for BimL to bind Bax but are important for it to efficiently activate Bax. Finally, BimL-dCTS functions only to bind and inhibit Bcl-XL. The defined mechanism(s) of these mutants makes them useful for probing the differential sensitivity of HEK293 and MEF cells to expression of VBimL-dCTS as seen in Figure 1. Expression of the mutants in HEK293 cells by transient transfection revealed that similar to VBimL-dCTS, expression of either VBimL-L129E or VBimL-I132E was not sufficient to kill HEK293 cells, despite expression of either mutant being sufficient to kill the primed MEF cell line (Figure 10). In contrast, HEK293 cells were killed by expression of VBimL-I125E, albeit to a lesser extent than by VBimL (Figure 10). This result is consistent with our findings with purified proteins showing that the EC50 for liposome permeabilization by BimL-I125E was 100 nM compared to ~1 nM for BimL (Figure 6B). The activity of VBimL-I125E also demonstrates that BimL binding to membranes is not required to kill HEK293 cells as BimL-I125E does not bind membranes (Figure 6B). Together, this data suggests that unlike MEF cells, only mutants of BimL that can efficiently activate Bax kill HEK293 cells.

          Discussion

          The apoptotic activity of Bim in live cells is likely mediated by a combination of functions that result in both activation of Bax and inhibition of anti-apoptotic proteins. Unlike any of the known BH3-proteins or small molecule inhibitors, BimL-dCTS inhibits all of the major multi-domain Bcl-2 family anti-apoptotic proteins without activating Bax or Bak. Thus expression of this tool protein in cells enables new insight into the importance of the extent to which a cell depends on the expression of anti-apoptotic proteins for survival (Figure 1A, Figure 2B). Our results strongly suggest that the varying levels of apoptotic response of cell lines to BimL-dCTS reflect the extent to which that particular cell type is primed. Thus, HEK293 cells and mature neurons that are resistant to inhibition of Bcl-2, Bcl-XL and Mcl-1 but sensitive to activation of Bax, are functionally unprimed. Partial resistance to expression of BimL-dCTS suggests that the flow of Bcl-2 family proteins between different binding partners leads to differential levels of dependency on the activation of Bax to trigger apoptosis. To illustrate this, we have created a schema illustrating protein flow at the two extremes represented by fully unprimed and primed cells and the effects of mutations in the Bim CTS on regulating apoptosis (Figure 11). In the schema, flow is indicated by the different lengths of the equilibria arrows and illustrates the consequences of the various dissociation constants displayed in Figure 6B. While BimL efficiently recruits Bax to membranes and activates it, BimL-I125E does not bind to membranes and has reduced binding to Bax therefore this mutant activates Bax less efficiently than BimL (Figure 11A). BH3-proteins that do not efficiently activate Bax, such as BimL-L129E or BimL-I132E, do not result in MOMP in unprimed cells (Figure 11A) instead they interact primarily with anti-apoptotic proteins (illustrated here as Bcl-XL since it was possible to measure binding with this purified protein). The binding measurements in Figure 6B allow prediction of the outcome of more subtle differences in interactions for BimL and its mutants. For example, even though BimL-I125E activates Bax the concentration required is around 100 nM while the dissociation constant for Bcl-XL is less than 3 nM (Figure 6B) such that in cells BimL-I125E would preferentially bind and inhibit Bcl-XL rather than activate Bax (Figure 11B). While the CTS is necessary for Bim to activate Bax at physiologically relevant concentrations, membrane binding mediated by the CTS is not a prerequisite for interaction with Bax. Rather, binding to membranes increases subsequent Bax activation possibly through facilitating Bax conformational changes on the membrane (Figure 6B compare BimL, BimL-CTS2A and BimL-I125E). Thus, it is likely that in cells expressing endogenous Bim, binding to membranes contributes to the efficiency with which the protein kills cells. In addition, a recent publication suggests that membrane-bound Bim is more pro-apoptotic as it can dimerize using the LC1-motif by binding to DLC1 (Singh et al., 2017). Here, we observed that one reason that BimL-dCTS is less pro-apoptotic is loss of binding to membranes where it can activate Bax more efficiently. Future work will determine the relative importance of the CTS-mediated Bim binding to mitochondrial membranes and direct activation of Bax. In addition, it will be critical to determine the role for the DLC1-mediated Bim complex formation on binding anti-apoptotic proteins in the membranes. A key question is whether the resulting alterations of Bim interactions with other Bcl-2 family proteins that are mediated by BH3-domain, the CTS and LC1 region are regulated differently in different cell types. Nevertheless, restoring membrane binding to BimL-dCTS using a mitochondrial tail-anchor (BimL-dCTS-MAO) did not restore Bax activation, thus indicating that the Bim CTS does more than bind the protein to membranes . Additionally, our work suggests a newly characterized mechanistic distinction between Bim and tBid, as tBid requires membrane binding and a subsequent conformational change in order to bind and efficiently activate Bax (Lovell et al., 2008; Shamas-Din et al., 2013a) while BimL can activate Bax in solution via dual interactions with the Bim BH3 domain and CTS (Figures 3C, 8, 9 and 11).

          The activities of the various Bim mutants analyzed here further suggest that specific residues in the Bim CTS enable physiological concentrations of Bim to activate Bax. That BimL-V124E, BimL-I125E and BimL-L129E all bind Bax in solution and in the presence of membranes with similar affinities yet vary functionally to trigger Bax-mediated liposome permeabilization by three orders of magnitude, suggests a specific role for this region in activation of Bax (Figure 6B) rather than the region simply increasing overall binding affinity. The situation is further complicated by another major role of the CTS of Bim in binding the protein to membranes. BimL-dCTS-MAO binds to mitochondria and liposomes yet is defective in activating Bax to permeabilize these membranes further suggesting a role for specific residues in the CTS binding to and activating Bax (Figure 7B). Such a role is consistent with our crosslinking data suggesting direct binding between the CTS of Bim and Bax (Figure 8) that is surprisingly mediated at least in part by the canonical BH3-binding groove in Bax (Figure 9). Furthermore, these CTS residues particularly L129 (which corresponds to L185 in BimEL) increased the affinity for Bim binding to Bcl-XL such that it conferred resistance to BH3 mimetic drugs (Liu et al., 2019). Nevertheless, it remains formally possible that changes in binding affinity coupled with alterations in effective off-rate due to membrane binding may also contribute to the activation of Bax by Bim. In addition to loss of interactions with the membrane BimL-dCTS still retains a greater propensity to activate Bax in comparison to Bim BH3-peptides (Compare Figure 3A with Sarosiek et al., 2013; Figure 4H). While 1 μM of BimL-dCTS was sufficient to activate Bax in our liposome release assay, over 10 μM of Bim BH3-peptide was required to achieve similar Bax activation. This result suggests that in addition to the BH3 domain and the CTS, other regions of Bim contribute to Bim’s pro-apoptotic function as previously suggested (Singh et al., 2017). Moreover, it is unclear to what extent other regions of Bim contribute to the BH3 region and the CTS adopting an optimal structure that can efficiently activate Bax. Similar to Bim, full length Bid cleaved by caspase is nearly 10-fold more active than a Bid-BH3 peptide in activating Bax, suggesting other regions outside the BH3-domain of Bid contribute to the interaction and/or activation of Bax. Thus, future studies of Bim and Bid should address what additional sequences other than the BH3-domains and C-terminal regions participate in Bax activation.

          Currently, BH3-profiling is the technique used to assay the state of apoptotic priming for different tissue types, however, this technique requires the addition of BH3-peptides at high concentrations, and can only be performed on cells/tissues after permeabilization of the plasma membrane (Potter and Letai, 2016). As an alternative, we propose lentiviral delivery and expression of BimL-dCTS be performed on living cells (or tissue samples), with readouts currently being used to assay cell death such as Annexin V staining, condensed nuclei, PI staining of nuclei, etc. Recently, it was reported in adults that most tissues are unprimed (Sarosiek et al., 2017); however, the status of priming for different cell types that make up a single tissue may differ. In contrast, in tissue culture most cells are at least partially primed (Figure 1). We speculate that stress responses that result when fully or partially transformed cell lines are grown under non-physiological conditions (high glucose and oxygen, in the presence of serum and on plastic with abnormal stromal interactions) generally account for the dependence of these cell lines on continued expression of anti-apoptotic proteins. BH3-profiling can only provide an answer at the tissue level or for cell populations that can be isolated in sufficient quantities or easily cultured (Sarosiek et al., 2017). However, lentiviral delivery and expression of VBimL-dCTS in cells in co- or organoid-cultures, tissue slices and in vivo can provide a means to assay the level of dependence of individual cells on expression of anti-apoptotic Bcl-2 family proteins. This information could prove valuable to understanding which cell types may be most affected by small molecule BH3 mimetics as chemotherapeutics and for other drugs to better predict and prevent off-target toxicities that result in cell priming.

          Overall, our data suggests a model in which the unusual CTS of Bim is not only required for binding to membranes but is directly involved in the activation of Bax. This interaction likely occurs via binding of the Bim CTS to the BH3-binding groove on Bax (Figure 9). However, the BH3-binding groove may not be the exclusive binding site for the Bim CTS. As seen in Figure 8C, the CTS of Bim photocrosslinks to 6H-Bax even in the presence of the L129E mutation. However, crosslinking is abolished though with the BH3 4E mutation. How Bim binding to the BH3 binding groove by both its BH3 region and CTS enables Bax activation is the subject of ongoing studies. Nevertheless, this function is crucial for BimL to kill unprimed cells. The CTS also increases the affinity of Bim for binding to Bcl-XL and Bcl-2 (Figure 11). The very much higher affinity of Bim for Bcl-XL and Bcl-2 compared to Bax ensures that in cells with excess anti-apoptotic proteins Bim is effectively sequestered and neutralized. In previous studies, we demonstrated that the additional affinity of the interaction of Bim with Bcl-XL and Bcl-2 provided by the Bim CTS is sufficient to dramatically reduce displacement of Bim by small molecule BH3 mimetics (Liu et al., 2019). Thus, regulation of apoptosis by Bcl-2 proteins is more complicated than presented in most current models. Moreover, the mutants and binding affinities described here provide the tools necessary for future studies of the relative importance of activation of Bax compared to inhibition of anti-apoptotic proteins in intact cells and in animals.

          Materials and methods

          Reagent type
          (species) or resource
          DesignationSource or referenceIdentifiersAdditional information
          Antibodyantibody to Cytochrome cIn house (Billen et al., 2008)(1:2000) Dilution
          AntibodyDonkey anti-rabbit (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-150(1:10000) Dilution
          AntibodyDonkey anti-mouse (polyclonal)Jackson Immuno Research LaboratoriesCat. #: 711-035-152(1:10000) Dilution
          AntibodyAntibody to BaxIn house (Zhu et al., 1996)Max6(1:1000) dilution
          AntibodyAntibody to BimSanta Cruz BiotechnologyCat. #: sc-11425(1:50) dilution
          Cell line (M. musculus)Baby Mouse Kidney(BMK)-DKO (Bax and Bak knockout) cellsOther (Degenhardt et al., 2002)Provided by Dr. Eileen White (Rutgers University)
          Mycoplasma free, see Materials and methods
          Cell line (H. sapiens)Cama-1RRID: CVCL_1115Provided by Dr. Linda Penn (University of Toronto). Mycoplasma Free, see Materials and methods
          Cell line (H. sapiens)HEK293Other (Graham et al., 1977)RRID: CVCL_0045Provided by Dr. Frank Graham (McMaster University).
          Mycoplasma Free
          , see Materials and methods
          Cell line (H. sapiens)HCT-116Other (Polyak et al., 1996)RRID: CVCL_0291Provided by Dr. Bert Vogelstein (John Hopkins University).
          Mycoplasma Free
          , see Materials and methods
          Strain (M. musculus)Embryonic day 15 embryosThe Jackson LaboratoryC57BL/6JUsed for the preparation of primary cortical neurons and for purification of mitochondria, see Materials and methods.
          Cell line (M. musculus)MEFOther (Pagliari et al., 2005)RRID: CVCL_U630Provided by Dr. Doug Green (St. Judes Children’s Research Hospital)
          Mycoplasma Free, see Materials and methods
          Chemical compound, drugDraq5ThermoFisher Scientific, Molecular probesCat. #62251Nuclear stain for live cell imaging
          Chemical compound, drugHoescht 33258Cell signaling technologiesCat. # 4082SNuclear stain for live cell imaging
          Chemical compound, drugTetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)ThermoFisher Scientific, Molecular probesCat. # T669Used to stain actively respiring mitochondria
          Chemical compound, drugPropidium iodideBioshopCat. # PPO888.10Nuclear stain for dead cells
          Chemical compound, drugAlexa 647-maleimideThermoFisher Scientific, Molecular probesCat. #: A20347Acceptor fluorophore in FRET experiments, when Alexa 568 is the donor.
          Chemical compound, drugAlexa568-maleimideThermoFisher Scientific, Molecular probesCat. #. A20341Donor fluorophore in FRET experiments when Alexa 647 is the acceptor.
          Chemical compound, drugANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt)ThermoFisher Scientific, Molecular probesA350Fluorophore used in liposome release assay (Billen et al., 2008)
          Chemical compound, drugDPX (p-Xylene-Bis-Pyridinium Bromide)ThermoFisher Scientific, Molecular probesX1525Quencher used in liposome release assay (Billen et al., 2008)
          Chemical compound, drugIANBD Amide (N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine)Molecular ProbesCat. #: D-2004Chemically reactive environment sensitive fluorophore. Reacts with Cysteine used to label proteins for in vitro study.
          Chemical compound, drugPC (L-α-phosphatidylcholine)Avanti Polar LipidsCat. #: 840051CFor making liposomes, used 48% PC
          Chemical compound, drugDOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine)Avanti Polar LipidsCat. #: 840035CFor making liposomes, used 10% DOPS
          Chemical compound, drugPI (L-α-phosphatidylinositol)Avanti Polar LipidsCat. #: 840042CFor making liposomes, used 10% PI
          Chemical compound, drugPE (L-α-phosphatidylethanolamine)Avanti Polar LipidsCat. #: 841118CFor making liposomes, used 28% PE
          Chemical compound, drugTOCL, (18:1 Cardiolipin)Avanti Polar LipidsCat. #: 710335CFor making liposomes, used 4% TOCL
          Chemical compound, drugbismaleimidohexane (BMH)Pierce22330Chemical crosslinker, Cysteine specific. Used for chemical crosslinking of Bim and Bax proteins, see Materials and methods.
          Commercial assay or kitFugene HDPromegaCat. #: E2311Transfection reagent for mammalian cells
          Commercial assay or kitTransIT-X2MirusCat. #: Mir 6003Transfection reagent for mammalian cells
          Gene (H. sapiens)BaxIn house (Yethon et al., 2003)GI: L22473.1Expression plasmid for production of recombinant protein
          Recombinant DNA reagent
          (H. sapiens)
          BaxIn house (Zhang et al., 2010; Zhang et al., 2016)GI: L22473.1For recombinant 6H-Bax protein used in photocrosslinking and for making Cys-null or single Cys recombinant protein in chemical crosslinking
          Recombinant DNA reagent (H. Sapiens)Bcl-XLIn house (Ding et al., 2014)GI: Z23115.1For recombinant 6H-Bcl-XL protein used in photocrosslinking, membrane permeabilization, and protein-protein binding assays.
          Gene (H. sapiens)BadIn house (Aranovich et al., 2012)GI: AB451254.1For expression of VBad in cells
          Gene (M. musculus)BidIn house (Lovell et al., 2008)GI: NM_007544.4For recombinant cBid purification (
          Gene (M. musculus)BimLThis paperGI: AAD26594.1This lab, plasmid # 2187, for recombinant BimL purificaton
          Recombinant DNA reagent
          (M. musculus)
          BimLThis paperGI: AAD26594.1Dr. Lin lab, plasmidpSPUTK-BimL For the single-Cys proteins used in photo and chemical crosslinking
          Recombinant DNA reagent
          (M. musculus)
          tBidIn house (Aranovich et al., 2012)GI: NM_007544.4For expression of VtBid in cells
          OtherCell Carrier-384, Ultra platePerkinElmerCat. #: 6057300For mono-layer culturing and imaging cell lines
          OtherGreiner Bio-one Cell culture microplate, 384 wellGreiner Bio-oneCat. 781090For culturing and imaging primary cortical neurons.
          OtherNon-binding surface, 96-well plate, black with clear bottomCorningCat. #: 3881For recombinant protein and liposome assays. It is critical to use a non-binding plate.
          OtherOpera PhenixPerkinElmerCat. #: HH14000000Automated confocal microscope. Used for imaging cell lines and primary cortical neurons.
          OtherAnnexinV*Alexa647In House (Logue et al., 2009)Used for detecting phosphotidylserine externalization (Blankenberg et al., 1998)
          OtherεANB-[14C]Lys-tRNALystRNA ProbesL-32Used for incorporation εANB-Lys into Bim protein using an in vitro translation system. The εANB-group is photoactive and generates a nitrene for photocrosslinking
          Other[35S]MethioninePerkinElmerNEG009CUsed for incorporation [35S]Met into Bim and Bax proteins using an in vitro translation system for photo or chemical crosslinking,see Materials and methods.
          Othertranscription/translation (TNT)-SP6 coupled wheat germ extract systemPromegaL4130Used for synthesis of Bim and Bax proteins for chemical crosslinking
          Othermulti-purpose image scannerFuji FilmFLA-9000Used for phosphorimaging to detect radioactive proteins in gels
          Software, algorithmGraphPad PrismSan Diego, CaliforniaVersion 6
          RRID:SCR_002798
          Scientific graphing program, used for curve fitting of in vitro data and to perform statistical analysis.
          Software, algorithmImageJPMID: 17936939MBF - ImageJ for microscopy, Dr. Tony Collins (McMaster University)For band density measurements used to quantify Cytochrome c release from immunoblots.
          Software, algorithmMulti GaugeFuji FilmVersion 3.0Used for processing and displaying phosphor-images
          Recombinant DNA reagent (A. victoria)mVenus-pEGFP-C1OtherGI: KU341334.1Dr. Ray Truant (McMaster University). Backbone EGFP-C1 (Clonetech)

          Protein purification

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          Wild type and single cysteine mutants of Bax, Bcl-XL, and cBid were purified as described previously (Kale et al., 2014). cBid mutant 1 (cBidmt1) was purified with the same protocol used for cBid (Kale et al., 2014). Bad was purified as described previously (Lovell et al., 2008). His-tagged Bax and Bcl-XL proteins were purified as described previously (Ding et al., 2014)

          His-tagged Noxa was expressed in E. coli strain BL21DE3 (Life Tech, Carlsbad, CA). E. coli cells were lysed by mechanical disruption with a French press. The cell lysate was diluted in lysis buffer (10 mM HEPES (7.2), 500 mM NaCl, 5 mM MgCl2, 0.5% CHAPS, 1 mM DTT, 5% glycerol, 20 mM Imidazole) and Noxa was purified by affinity chromatography on a Nickel-NTA column (Qiagen, Valencia, CA). Noxa was eluted with a buffer containing 10 mM HEPES (7.2), 300 mM NaCl, 0.3% CHAPS, 20% glycerol, 100 mM imidazole, dialyzed against 10 mM HEPES 7.2, 300 mM NaCl, 10% glycerol, flash-frozen and stored at −80°C.

          Purification of BimL and single cysteine mutants of BimL was carried out as previously described (Liu et al., 2019). Briefly, cDNA encoding full-length wild-type murine BimL was introduced into pBluescript II KS(+) vector (Stratagene, Santa Clara, CA). Sequences encoding a polyhistidine tag followed by a TEV protease recognition site (MHHHHHHGGSGGTGGSENLYFQGT) were added to create an in frame fusion to the N-terminus of BimL. All the purified BimL proteins used here retained this tag at the amino-terminus. However, control experiments demonstrated equivalent activity of the proteins before and after cleavage with TEV protease (Data not shown). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis.

          BimL was expressed in Arabinose Induced (AI) E. coli strain (Life Tech, Carlsbad, CA). E. coli were lysed by mechanical disruption with a French press. Proteins were purified from the cell lysate by affinity chromatography using a Nickel-NTA column (Qiagen, Valencia, CA), and eluted with a solution containing 20 mM HEPES pH7.2, 10 mM NaCl, 0.3% CHAPS, 300 mM imidazole, 20% Glycerol. The eluate was adjusted to 150 mM NaCl and applied to a High Performance Phenyl Sepharose (HPPS) column. Bim was eluted with a no salt buffer and dialyzed against 10 mM HEPES pH7.0, 20% glycerol, flash-frozen and stored at −80°C.

          Protein labeling

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          Single cysteine mutants of Bax, Bcl-XL, cBid and Bad were labeled with the indicated maleimide-linked fluorescent dyes as described previously (Pogmore et al., 2016; Kale et al., 2014; Lovell et al., 2008). Single cysteine mutants of Bim were labeled with the same protocol as cBid with the exception that the labeling buffer also contained 4M urea.

          Bim binding to membranes

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          Liposomes (100 nm diameter) with a lipid composition resembling MOM were prepared as described previously (Kale et al., 2014). Mouse liver mitochondria were isolated from Bak-/-mice as previously described (Pogmore et al., 2016). Liposomes and mitochondria were labeled with 0.5% and 2% mass ratios of DiD, respectively (Life Tech, Carlsbad, CA). The single-cysteine mutant of BimL, BimL Q41C, was labeled with Alexa568-maleimide and incubated with the indicated amount of unlabeled or DiD-labeled mitochondria or liposomes at 37°C for 1 hr. Intensities of Alexa568 fluorescence were measured in both samples as Funlabeled and Flabelled respectively using the Tecan infinite M1000 microplate reader. FRET, indicating protein-membrane interaction, was observed by the decrease of Alexa568 fluorescence when BimL bound to DiD labeled membranes compared to unlabeled membranes. FRET efficiency was calculated as described previously (Shamas-Din et al., 2013a). The data was fit to a binding model as described below. Lines of best fit were calculated using least squares in Graphpad Prism software.

          Calculating the number of Bim molecules per Liposome

          Step 1: Find the total number of Bim molecules in a 2 mL reaction

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          Concentration of Bim: 5 nM in 2 mL reaction

          Number of Bim (mole) = concentration (M) x volume (L)

          • = 5×10−9 M x 0.002 L

          • = 1×10−11 mol

          Total number of Bim molecules in 2 mL reaction = Number of Bim (mole) x Avogadro’s Constant

          • = 1×10−11 mol x 6.02 × 1023 mol−1

          • = 6.02×1012 Bim molecules

          Step 2: Find the total lipid surface area or the total number of liposome made from 1 mg lipid film

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          The total number of lipid molecules in a liposome are given by the formula below

          Ntotal = [4π(100 nm/2)2+4π((100 nm/2)–5) 2]/0.71nm2 = 80088.49 lipid molecules

          Assuming that the average area of the lipid head group in our liposome is 0.71 nm2 (because it is made out of mostly PC).

          Total lipid in 1 mg lipid film (in mole)=mass in gram/molecular wt (M.W) in gmol−1

          • = 0.001 g/ 804.95 gmol−1

          • = 1.24×10−6 mole of lipid

          Total lipid molecules in 1 mg lipid film = 1.24×10−6 mol x 6.02 × 1023 mol−1

          • = 7.48 × 1017 lipid molecules

          Molecular weight of our mitochondrial-like lipid film was calculated from the %Molar and molecular weight of individual lipid which was published in Kale et al. (2014) (Examining the Molecular Mechanism of Bcl-2 Family Proteins at Membranes by Fluorescence Spectroscopy).

          Total number of liposomes = Total lipid molecule in 1 mg/Ntotal = 9.34 × 1012 liposomes.

          Surface area of a liposome = 4πr2 = 4π(50 nm)2 = 10000π nm2

          where r is the radius of our liposome.

          Total lipid surface area = surface area of a liposome x total number of liposome = 2.93×1017 nm2

          Step 3: Calculate the number of Bim per liposome or per surface area

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          Number of Bim per liposome = Total number of Bim in a 2 mL reaction/Total number of liposome

          • = 6.02 × 1012 Bim molecules/9.34 × 1012 liposomes=0.64 Bim molecule per liposome

          Number of Bim per surface area = Total number of Bim in a 2 mL reaction/Total lipid surface area

          • = 6.02 × 1012 Bim molecules/2.93 × 1017 nm2=2.05×10−5 Bim molecule per nm2

          Membrane permeabilization

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          Membrane permeabilization assays with liposomes encapsulating ANTS and DPX were performed as described previously (Kale et al., 2014). To measure permeabilization of BMK mitochondria, the indicated amounts of proteins were incubated with mitochondria (1 mg/mL) purified from BMK cells genetically deficient for Bax and Bak expressing mCherry fluorescent protein fused to the SMAC import peptide responsible for localization in the inter-membrane space. After incubation for 45 min at 37°C samples were centrifuged at 13,000 g for 10 min to separate the pellet and supernatant fractions and membrane permeabilization was calculated based on the mCherry fluorescence in each fraction (Shamas-Din et al., 2014). For mouse liver mitochondria, cytochrome c release was measured by immunoblotting as described previously (Pogmore et al., 2016; Sarosiek et al., 2013).

          BH3 profiling

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          Heavy membranes enriched in mitochondria were isolated as described previously (Pogmore et al., 2016; Brahmbhatt et al., 2016). Membrane fractions (1 mg/mL) were incubated with 500 nM of the specified BH3-proteins (Bim, Bad and/or Noxa). For E15 brain mitochondria, 0.5 mg/mL of membrane fractions were used and incubated with the indicated amounts of BH3-only proteins for 30 min at 37 °C. Membranes were pelleted by centrifugation at 13,000 g for 10 min and cytochrome c release was analyzed by immunoblotting using a sheep anti-cytochrome c antibody (Capralogics). Mitochondria from embryonic mouse brains for BH3profiling experiments were prepared from ~20 mouse embryos, E15 in age, following the same protocol used for liver mitochondria (Pogmore et al., 2016).

          Protein-protein binding

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          For FRET experiments, single cysteine mutants of cBid (126C), Bcl-XL (152C), Bax (126C), BimL (41C) and BimL mutants were purified and labeled with either Alexa 568-maleimide (donor) or Alexa 647-maleimide (acceptor) as specified. To determine binding a constant amount of donor protein was incubated with the indicated range of acceptor proteins and where specified liposomes or mitochondria. The intensity of Alexa568 fluorescence with unlabeled or Alexa647-labeled Bcl-XL was measured as Funlabeled or Flabeled, respectively, and FRET was calculated as described in Pogmore et al. (2016). All measurements were collected using the Tecan infinite M1000 microplate reader. Lines of best fit were calculated using least squares in Graphpad Prism software.

          For each pair of proteins a dissociation constant (Kd) was measured in solution and with liposomes. Curves were fit to an advanced function taking into account change of the concentration of acceptor ([A]) when [A] is close to Kd:

          [D] is the concentration of donor, F indicates the FRET efficiency with the concentration of acceptor as [A], Fmax is the maximum FRET efficiency in the curve (Pogmore et al., 2016).

          Photo and chemical crosslinking of Bim to Bax or Bcl-XL

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          To produce the proteins for crosslinking using in vitro systems, the DNA sequence encoding murine BimL Cys-null and Lys-null mutant without the His tag and TEV protease recognition site was excised from the pBluescript II KS(+) vector by restriction endonucleases NcoI and ClaI and inserted into the pSPUTK vector (Stratagene, Santa Clara, CA). Mutations as specified in the text were introduced into this sequence using site-directed mutagenesis to generate the single-Lys BimL or single-Cys mutants.

          The photocrosslinking method for studying interactions among the Bcl-2 family proteins has been described in detail (Lin et al., 2019). Briefly, using the RNAs produced from the single-Lys Bim DNAs in the pSPUTK vector by an in vitro transcription system, [35S]Met-labeled BimL proteins with a single εANB-Lys incorporated at specific locations were synthesized in an in vitro translation system. 10 μL of the resulting BimL proteins were incubated at 37 °C for 1 hr with 1 μM of 6H-Bax or 6H-Bcl-XL protein and Bak-/-mouse liver mitochondria (0.5 mg/ml total protein and were resuspended in AT buffer with 80 mM KCl and supplemented with energy regenerating system as described previously Yamaguchi et al., 2007) in a 21 μL reaction adjusted by buffer A (110 mM KOAc, 1 mM Mg(OAc)2, 25 mM HEPES, pH 7.5). The mitochondrial and soluble fractions were separated by centrifugation at 13,000 g and 4 °C for 5 min, and the mitochondria were resuspended in 21 μL of buffer A. Both mitochondrial and soluble fractions were photolyzed to induce crosslinking via the ANB probe. The resulting samples were adjusted to 250 μL with buffer B (buffer A with 1% Triton X-100 and 10 mM imidazole) and incubated with 10 μL of Ni2+-chelating agarose at 4 °C for overnight. After washing the Ni2+-beads three times with 350 μL of buffer B and one time with 400 μL of PBS, the photoadducts of the radioactive BimL protein and the 6H-tagged Bax or Bcl-XL protein and other proteins bound to the Ni2+-beads were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphor-imaging.

          For chemical crosslinking, [35S]Met-labeled single-Cys or Cys-null BimL and Bax proteins were synthesized from the respective mutant Bim and Bax DNAs in the pSPUTK vector using a transcription/translation (TNT)-coupled in vitro system (Promega, Madison, WI). The resulting BimL and Bax proteins, 2 μL each, were paired as indicated in Figure 9, and reduced by 11 μM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a 20 μL reaction adjusted with buffer A at 37 °C for 1 hr. The sample was diluted to 110 μL with buffer A and split evenly to two aliquots. For a ‘60 min’ BMH crosslinking reaction, one aliquot was incubated with 0.1 mM BMH and 6 mM EDTA at 25 °C for 60 min, and the reaction was stopped by incubation with 50 mM 2-mercaptoethanol at 25 °C for 15 min. For a ‘0 min’ control reaction, the other aliquot was incubated with 2-mecaptoethanol and EDTA for 15 min, and then with BMH for 60 min. The resulting samples were precipitated by trichloroacetic acid. The resulting protein pellets were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

          To obtain the immunoprecipitation data in Figure 9—figure supplement 1, the indicated single-Cys BimL and Bax proteins produced by the TNT system, 4 μL each, were paired and reduced by TCEP. The sample was diluted to 260 μL with buffer A and crosslinked by BMH. The resulting sample was divided to two aliquots. The 85 μL or 170 μL aliquot was immunoprecipitated by Bax or Bim antibody, respectively. Thus, each aliquot was adjusted to 250 μL with IP buffer (100 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 1 mM PMSF, 1% (v/v) Triton X-100), and received Bax antibody (made in house, 1:1000 dilution) or Bim antibody (Santa Cruz Biotechnology, Dallas, TX, 1:50 dilution). The samples were rotated at 4 °C for overnight, and after receiving 25 μL of Protein G Sepharose (50% suspension in IP buffer), rotated for 2 more hours. After centrifugation at 2000 g for 0.5 min, the beads were washed three times with 400 μL of IP buffer and one time with 400 μL of 100 mM Tris pH 7.5 and 100 mM NaCl. The bound proteins were eluted with reducing SDS sample buffer and analyzed by SDS-PAGE and phosphorimaging.

          Measurement of cell death in response to expression of VBimL constructs

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          HEK293, BMK, MEF, and HCT116 cells were maintained at 37°C (5% v/v CO2) in dMEM complete [dMEM, 10% Fetal Bovine Serum, 1% essential amino acids (Gibco, Grand Island, NY)]. CAMA-1 were maintained the same environmental conditions but using dMEM/F12 (Gibco, Grand Island, NY). Cell lines were routinely confirmed to be mycoplasma-free using a PCR-based protocol as described by Hopert et al. (1993), and their authenticity was verified by short-tandem repeat (STR) profiling at The Centre for Applied Genomics (Toronto, ON, Canada) for human cells. Murine cell lines have not been authenticated. Cells were seeded in CellCarrier-Ultra 384-well plates (1000 cells/well for BMK and MEF, 2000 cells/well for HEK293 and HCT116, 3000 cells/well for CAMA-1). One day later, cells were transfected using FugeneHD (Promega, Madison, WI) with plasmids encoding Venus, or Venus-fused BimL constructs in an EGFP-C3 backbone. Cell culture medium was added to each reaction (50 µl/0.05 μg DNA) and the whole mix added to each well (50 µl/well) of a pre-aspirated 384-well plate of cells. After 24 hr, cells were stained with Draq5 and Rhodamine-labeled Annexin V and image acquisition was performed using the Opera QEHS confocal microscope (Perkin Elmer, Woodbridge, ON) with a 20x air objective. Untransfected cells and cells treated with 1 µg/mL staurosporine were used as negative and positive controls for Annexin V staining. Cells were identified automatically using software as described previously (Shamas-Din et al., 2013a). Intensity features were extracted using a script (dwalab.ca) written for Acapella high content imaging and analysis software (Perkin Elmer, Woodbridge, ON). Cells were scored as Venus or Annexin V positive if the Venus or Annexin V intensity was greater than the average intensity plus two standard deviations for the Venus or Annexin V channels in images of non-transfected cells. Cell death ascribed to the VBimL fusion proteins was quantified as the percentage of Venus-positive cells that were also Annexin V positive. For neuron cultures, cell segmentation using conventional methods could not be achieved due to complex cellular morphologies. Therefore, nuclei were first identified, then a ring region ~10% of nuclear area was drawn around each nucleus. Venus intensity was calculated for this ring region, representing the neuronal cell body, to determine if the neuron was expressing the Venus fluorescent protein.

          Primary neuron cultures

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          Primary cortical neuron cultures were prepared from embryonic day 15, C57BL/6J mouse embryos as previously described (Mergenthaler et al., 2012). All animal breeding and handling were performed in accordance with local regulations and after approval by the Animal Care Committee at Sunnybrook Research Institute, Toronto. Briefly, after separation from hippocampus and subcortical structures, cortices were washed twice with ice-cold PBS, digested with 1x trypsin for 15 min at 37°C, washed twice with ice-cold PBS and then resuspended with a flame-treated glass pipette in N-Medium (DMEM, 10% v/v FBS, 2 mM L-glutamine, 10 mM Hepes, 45 µM glucose). The dissociated cortices were gently pelleted by centrifugation (200 g for 5 min), N-media was removed, and neurons were resuspended and cultured in Neurobasal-Plus medium (ThermoFisher Scientific) supplemented with B27-Plus (ThermoFisher Scientific) and 1x Glutamax (ThermoFisher Scientific). Neurons were seeded at 5000 cells per well in a 384 well plate (Greiner µclear) after coating with poly-d-lysine (Cultrex). The medium was partially replaced on day 5 in culture with Neurobasal-Plus supplemented with B27-Plus and 1x Glutamax.

          Lentivirus to express VBimL and other BimL mutants were cloned into the pTet-O-Ngn2-Puro construct with the Ngn2 gene cut out. This construct was a kind gift from Dr. Philipp Mergenthaler, Charité Universitätsmedizin Berlin. Primary neuron cultures were infected with both VBimL and rtTA lentiviral particles (~3 μL of each concentrated stock) on the day of seeding. 24 hr later, Neurobasal-Plus medium containing lentiviral particles was removed and replaced with fresh Neurobasal-Plus medium.

          Doxycyline (ThermoFisher scientific) was added to 16 day in vitro old cultures of neurons at a concentration of 2 μg/mL to induce VBimL protein expression. 5 hr later, neurons were stained with 5 µM Draq5 (Thermofisher scientific) and 0.1 μM TMRE (Thermofisher scientific), then incubated for 30 min at 37 °C. Confocal microscopy was performed immediately after.

          Lentiviral production

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          Each lentivirus was made using the following protocol adhering to biosafety level two procedures. On day 0, lentiviral vectors psPax2 (10 μg) and pMD2.G (1.25 μg) were mixed with 10 μg of desired VBimL lentiviral construct in 1000 μL of Opti-MEM media (ThermoFisher Scientific). Next, 42 μL of polyethylenimine (PEI) solution [1 mg/mL] was added, the mixture vortexed, then allowed to settle for 15 min at room temperature. After 15 min, 1.5 × 107 of resuspended HEK293 cells and the transfection solution were mixed and seeded onto a 100 mm culture dish with 10 mL of dMEM complete plus 10 μM of the caspase inhibitor Q-VD-Oph (Selleckchem), and left to incubate at 37 °C (5% v/v CO2) for 72 hr. On day 3, media containing lentiviral particles was filter sterilized using a 0.45 μm polyethersulfone filter, and mixed with polyethylene glycol (Bioshop) to achieve a final concentration of 10% (w/v). This was left to mix and precipitate the virus overnight at 4 °C. On day 4, the media was centrifuged for 1 hr at 1600 g, supernatant was then removed and the pellet was resuspended with 400 μL of Neurobasal-Plus media (no additives). Resuspended virus was then stored at −80 °C until needed.

          Data availability

          Data generated or analysed during this study are included in the manuscript and supporting files.

          References

          Источник: https://elifesciences.org/articles/44525

          The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

          Abstract

          When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

          Introduction

          Most intracellular functions are carried out by proteins, assembled as chains of peptide-bond linked amino acid (aa) residues on large ribonucleoprotein particles called ribosomes. The aa-sequences are specified by information stored as deoxyribonucleic acid (DNA) sequences in the genome and transcribed into sequences of messenger RNAs (mRNAs). The mRNAs are translated into aa-sequences with the help of transfer RNAs (tRNAs) reading any of their 61 aa-encoding ribonucleotide triplets (codons). In termination of translation, the complete protein is released from the ribosome by a class-1 release factor (RF) recognizing one of the universal stop codons (UAA, UAG, and UGA), signaling the end of the amino acid encoding open reading frame (ORF) of the mRNA. There are two RFs in bacteria, RF1 and RF2, one in eukarya, eRF1. RF1 and RF2 read UAA, UAG, and UAA, UGA, respectively, while the omnipotent eRF1 reads all three stop codons. Each stop codon in the decoding center (DC) is recognized by a stop-codon recognition (SCR) motif in a class-1 RF, and all RFs have a peptidyl transfer center (PTC)-binding GGQ motif, named after its universal Gly–Gly–Gln triplet (GGQ), for coordinated ester bond hydrolysis in the P-site bound peptidyl-tRNA. The crystal structures of free RF1 and RF2 have a distance between the SCR and GGQ motifs of about 20 Å1,2, much shorter than the 70 Å separating DC and PTC in the bacterial 70S ribosome. This distance discrepancy made the expected coordination between SCR and ester bond hydrolysis enigmatic. The crystal structure of free eRF1 has, in contrast, about 70 Å between its SCR and GGQ motifs, a distance close to the 80 Å between the DC and PTC of the 80S ribosome in eukarya3. Further cryo-EM work showed that ribosome-bound RF1 and RF2 have extended structures4,5, facilitating coordinated codon recognition in DC and ester bond hydrolysis in PTC. Subsequent high-resolution X-ray crystal6,7,8,9,10,11,12,13 and cryo-EM14,15,16,17,18,19 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF220, eRF121, and GGQ-mediated ester bond hydrolysis22.

          If the compact forms of free RFs in the crystal1,2 are physiologically relevant, it would mean that eubacterial RFs are in the compact form upon A-site entry (pre-accommodation state) and assume the extended form (accommodation state) in a stop-codon dependent manner. The relevance is indicated by a compact crystal structure of RF1 in a functional complex with its GGQ-modifying methyltransferase23,24, although SAXS data indicated free RF1 to be extended in bulk solution25. At the same time, SAXS data from T. thermophilus RF2 free in solution suggested a compact form for the factor or, possibly a mixture of compact and extended forms26. The existence of a RF-switch from a compact, free form to an extended ribosome-bound from would make high-resolution structures of these RF-forms necessary for a correct description of the stop-codon recognition process, hitherto based on post-termination ribosomal complexes27,28.

          Indirect evidence for rapid conformational activation of RF1 and RF2 after A-site binding has been provided by quench-flow based kinetics22, and in a series of recent FRET experiments Joseph and collaborators showed free RF1 to be in a compact form29, compatible with the crystal forms of RF11 and RF22, but in an extended form when bound to the A site of the stop-codon programmed ribosome29. Ribosome-bound class-1 RFs in the compact form has been observed together with alternative ribosome-rescue factor A (ArfA) in ribosomal rescue complexes, which lack any codon in the A site14,15. Very recently, Svidritskiy and Korostelev6 used X-ray crystallography in conjunction with the peptidyl transfer-inhibiting antibiotic blasticidin S (BlaS) to capture a mutated, hyper-accurate variant of RF1 in the stop codon-programmed termination complex. They found RF1 in a compact form, which they used to discuss stop-codon recognition in conjunction with large conformational changes of the RFs. It seems, however, that this BlaS-halted ribosomal complex is in a post-recognition state (i.e., stop-codon recognition motif has the same conformation as in the post-accommodation state in DC) but before RF-accommodation in the A site, making its relevance for on-pathway stop-codon recognition unclear (However, see also below!).

          Here, in contrast, we use time-resolved cryo-EM30,31,32,33,34 for real-time monitoring of how RF1 and RF2 ensembles change from compact to extended RF conformation in the first 100 ms after RF-binding to the pre-termination ribosome. These compact RF-structures, originating from short-lived ribosomal complexes previously out of reach for structural analysis, are seen at near-atomic resolution (3.5–4 Å). The time-dependent ensemble changes agree qualitatively with accompanying and previous22 quench-flow studies. We discuss the role of the compact structures of RF1 and RF2 for fast and accurate stop-codon recognition in translation termination.

          Results

          Kinetics study predicts compact RF1/RF2 exist at 20 ms

          We assembled a UAA-programmed release complex, RC0, with tripeptidyl-tRNA in the P site, and visualized its structure with cryo-EM (Methods and Supplementary Fig. 1). The RC0 displays no intersubunit rotation, and the tripeptide of its P-site tRNA is seen near the end of the peptide exit tunnel (Supplementary Fig. 1). The mRNA of the DC is disordered, but the overall resolution of the RC0 is high (2.9 Å). Apart from a small fraction of isolated ribosomal 50S subunits, the RC0 ensemble is homogeneous (Supplementary Fig. 1). We used quench-flow techniques to monitor the time evolution of the class-1 RF-dependent release of tripeptide from the peptidyl-tRNA with UAA-codon in the A site after rapid mixing of RC0 with RF1 or RF2 at rate-saturating concentrations (kcat-range)22 (Fig. 1a). The experiments were performed at pH values from 6 to 8 units, corresponding to [OH] variation in the 0.25–2.5 µM range (Supplementary Figs 2 and  3). The results are consistent with the existence of a two-step mechanism, in which a pH-independent conformational change (rate constant kconf) is followed by pH-dependent ester bond hydrolysis (see Methods). We estimate kconf as 18 ± 3 s−1 for RF1 and 11 ± 1 s−1 for RF2 at 25 °C, which approximates the effective incubation temperature for the time-resolved cryo-EM experiments (Supplementary Figs 2 and  3).

          Time evolution of ribosome ensembles in termination of translation a. Cartoon visualization of the pathway from free release complex to peptide release. Compact class 1 release factor (RF) binds to RF-free ribosomal release complex (RC0) and forms the RC·RFcompact complex with compounded rate constant ka·[RFfree]. Stop codon recognition induces conformational change in the RF which brings the ribosome from the RC·RFcompact to the RC·RFextended complex with rate constant kconf. The ester bond between the peptide and the P-site tRNA is hydrolyzed with rate constant khydr. b Predicted dynamics of peptide release with conformational change in RF1. We solved the ordinary differential equations associated with termination according to the scheme in a with association rate constant ka = 45 µM−1s−1, [RF1free] = 3 µM, kconf = 18 s−1 and khydr = 2 s−1 (Supplementary Fig. 2) and plotted the fractions of ribosomes in RC0, RC·RFcompact and RC·RFextended forms (y-axis) as functions of time (x-axis). Green dot lines, RC0; red solid lines, RC·RFcompact; blue dash lines, RC·RFextended. c Predicted dynamics of peptide release with conformational change in RF2. The fractions of ribosomes in different release complexes were obtained in the same way as b with the rate constants ka = 17 µM−1 s−1, [RF2free] = 3 µM, kconf = 11 s−1 and khydr = 2.7 s−1 (Supplementary Fig. 3). d, e The populations of release complexes containing compact conformation and extended conformation of RF1 (d) and RF2 (e) at the 24 ms, 60 ms and long incubation time points as obtained by time-resolved cryo-EM after 3D classification of the particle images

          Full size image

          From the quench-flow data, we predicted that the ensemble fraction of the compact RF1/2 form would be predominant at 24 ms, and much smaller at 60 ms (Fig. 1b, c). These predictions are in qualitative agreement with the time-resolved cryo-EM data, which show a somewhat faster conformational transition than in the quench-flow experiments (Fig. 1d, e). The difference in termination rates is, we suggest, due to a local temperature increase by friction inside the microfluidic chip. We first focus on the cryo-EM structures of RF1, and then highlight the few structural differences between RF1 and RF2.

          Time-resolved cryo-EM analysis

          At 24 ms reaction time, 25% of ribosome-bound RF1 is in the compact form in what we name the pre-accommodation state of the ribosome (Fig. 1d). The 70S part of the complex is similar to that of the pre-termination complex preceding RF-binding, but there is an additional A-site density belonging to RF1 (Fig. 2a). In pre-accommodation state of the ribosome, domain III of RF1 is 60–70 Å away from the PTC, in a similar relative orientation as in the crystal forms of the free factors1,2 (Supplementary Fig. 4) and significantly differing from that in the post-accommodated state of the terminating ribosome5. The loop that contains the GGQ motif of RF1 is positioned at the side of the β-sheet of domain II (near aa 165–168), facing the anticodon-stem loop and the D stem of the P-site tRNA (Fig. 2a, c).

          Cryo-EM structures of E.coli 70S ribosome bound with release factor 1. a Pre-accommodated ribosome complex bound with RF1 in a compact conformation. b Accommodated ribosome complex bound with RF1 in an extended conformation. Light blue: 50S large subunit; light gold: 30S small subunit; green: tripeptide; orange: P-tRNA; pink: mRNA; red: compact RF1; and dark blue: extended RF1. c, d Positions of domain III of ribosome-bound RF1 in pre-accommodated ribosome complex (c) and accommodated RF1-ribosome complex (d) relative to mRNA (pink), P-tRNA (orange) and tripeptide (green). e, f Close-up views of the upper peptide exit tunnel, showing tripeptide (green) in pre-accommodated ribosome complex (e) and accommodated ribosome complex (f)

          Full size image

          At 60 ms reaction time the RF1-bound ribosome ensemble is dominated by the extended form of RF1 (Fig. 2b, d). We term the ribosome complex with extended RF1 the accommodated RF1-ribosome complex. It contains density for the tripeptide in the exit tunnel, indicating that at 60 ms the peptide has not been released from the ribosome (Fig. 2e, f and Supplementary Fig. 5). At a much later time-point (45 s) the tripeptide density is no longer present in the exit tunnel of the accommodated RF-ribosome complex. Precise estimation of the time evolution of tripeptide dissociation from the ribosome would require additional time points. Of particular functional relevance would be estimates of the time of dissociation of longer peptide chains from the exit tunnel.

          The most striking difference between the compact and extended conformation of ribosome-bound RF1 is the position of the GGQ of domain III. As RF1 switches its conformation from the compact to the extended form, the repositioning of domain III places the catalytic GGQ motif within the PTC, and adjacent to the CCA end of the P-site tRNA (Fig. 2c, d). The extended form of RF1 has a similar conformation as found in the previous studies7,10,12,13,35,36.

          Similar to the case of sense-codon recognition by tRNA, three universally conserved DC residues, A1492, A1493, and G530 of the ribosome’s 16S rRNA undergo key structural rearrangements during translation termination. In the RF-lacking termination complex, A1492 of helix 44 in 16S rRNA stacks with A1913 of H69. A1493 is flipped out and stabilizes the first two bases in the A-site stop codon. G530 stacks with the third base A in the stop codon. In the presence of RF, whether compact or extended, A1492 is flipped out towards G530 and interacts with the first two stop-codon bases. A1493 stacks with A1913, which is in close contact with A1492 in the RF-lacking termination complex. G530 stacks with the third stop-codon base (Fig. 3a, b).

          Interaction of RF1 with the ribosomal decoding center. a, b Structures of the ribosomal decoding center in pre-accommodated ribosome complex (a) and accommodated ribosome complex (b). Red: compact RF1; dark blue: extended RF1. c, d Conformations of switch loop in pre-accommodated ribosome complex (c) and accommodated ribosome complex (d). Gold: A1492 and A1493; and orange: S12

          Full size image

          The switch loop, which was previously proposed to be involved in inducing a conformational change of RF110,36, shows no interaction with protein S12 or 16S rRNA in the compact form of RF1 (Fig. 3c) whereas in the extended form of RF1, the rearranged conformation of the switch loop is stabilized by interactions within a pocket formed by protein S12, the loop of 16S rRNA, the β-sheet of domain II, and A1493 and A1913 (Fig. 3b, d). Shortening the switch loop (302–304) resulted in a substantially slower, rate-limiting step in peptide release37, which indicates that the switch loop plays a role in triggering the conformational change of RF1.

          Similar experiments were carried out for RF2 at 24 ms, 60 ms and 5 h reaction times. RF2 undergoes a conformational change from compact to expanded form similar to that of RF1 (Fig. 1e). As in the case of RF1, the switch loop of RF2 makes no contact with protein S12 or 16S rRNA. In the extended form of RF2, A1492 is flipped out from helix 44 (h44) of 16S rRNA and stacks on the conserved Trp319 of the switch loop, stabilizing the extended conformation of RF2 on the ribosome.

          The ribosome complexes with RF1/2 bound in compact conformation seen here are distinct from those reported for the ribosome rescue complex, in which ArfA is bound in the A site lacking a stop codon14,15. In our structures, the conformation of the conserved 16S rRNA residues in the DC (A1492, A1493, and G530) is similar to the classical termination configuration10. In contrast, in the presence of ArfA, these residues adopt conformations known from sense-codon recognition14,15. It suggests that the compact RFs bind to the A site regardless of the conformation of the DC. The conformational change of RFs is likely due to the changes in the switch loop triggered by its interaction with protein S12 and 16S rRNA. This interaction is disrupted by the mutation A18T of ArfA, hence leaving RFs in the compact conformation15.

          Our ribosome complexes with RF1/2 are also distinct from a recent ribosome complex with compact RF1, reported by Svidritskiy and Korostelev6. Shortening of the switch loop, combined with the addition of the antibiotic BlaS which prevents the GGQ motif from reaching the PTC, stabilizes ribosome-bound RF1 in a compact conformation6, distinct from the transient, compact RF1-structure observed here. In our structure, the SCR between the β4–β5 strands on domain II are bound loosely to the A site (Fig. 3a). In the BlaS-halted compact RF1 structure6, in contrast, the stop codon-recognition motif of RF1 has moved further into the A site by 5 Å, to a position almost identical to that of the fully accommodated, extended structure of RF1. The functional role of their structure is not immediately obvious, but if it can be interpreted as an authentic transition state analogue, the roles of our respective complexes would be complementary. We previously found that high accuracy of stop signal recognition depends on smaller dissociation constant (Km-effect) and larger catalytic rate constant (kcat-effect) for class-1 RF reading of cognate stop codons compared to near-cognate sense codons38. The Km-effect contributes by factors from 100 to 3000 and the kcat-effect by factors from 2 to 3000 to the overall termination accuracy values in the 103–106 range38. Accordingly, the present structure may represent binding of RFs in a transient state where rapid and codon-selective dissociation rates are responsible for the accuracy factor due to the Km-effect. Furthermore, Korostelev's structure6, with its comparatively deep interaction between the cognate stop codon and SCR center, could mimic the authentic transition state on the path from compact to the extended form of the RF. Accordingly, Korostelev's structure may illustrate additional selectivity due to the kcat-effect. To test these hypotheses, molecular computations28 based on our respective RF structures could be used to compare their stop codon selectivities with those of RFs in the post-termination state of the ribosome20.

          In a recent paper on the role of RF3 in the dissociation of the release factors RF1 and RF239, the authors observed an interaction between domain I of RF1 and L7/L12 proteins, which assists the binding of RF1, as supported by complementary functional analysis using L7/L12 deletion mutants. However, such an interaction is not observed in our structures. Another recently published study using smFRET40 reported two states of the termination complex, non-rotated and rotated, in apparent contradiction to our results as we only found one, non-rotated state. The rotated-state subpopulation observed by Adio et al.40 may represent the post-termination ribosome unbound to RF1/RF2, as also suggested by previous single-molecule work from Puglisi and Gonzalez labs41,42.

          Discussion

          During translation termination, the release of the nascent peptide must be strictly coordinated with the recognition of a stop codon at the A site. Our cryo-EM analysis shows that in the presence of a class-1 RF the bacterial ribosome adopts several states. After rapid addition of RF1 or RF2 to a ribosomal termination complex with tripeptidyl-tRNA attached at the P site, we first observe the pre-accommodated RF-ribosome complex (compact form of RF) at 24 ms with the peptide attached to the P-site tRNA. This, we suggest, is the first step in the termination reaction. Second, at 60 ms, we observe the accommodated RF-ribosome complex with the extended form of RF and the tripeptide in the exit tunnel. Third, at a much later time point, we observe the post-accommodated RF-ribosome complex, with the extended form of RF without tripeptide in the exit tunnel (Supplementary Fig. 5). These pieces of evidence from our time-resolved experiments clearly reflect the sequence of events in termination of bacterial protein synthesis. A structure-based model for the stepwise interaction between ribosome and RF and the release of the nascent peptide from the termination complex during the translation termination process is presented in Fig. 4. It shows how the ribosome traverses (1) the pre-termination state with the stop codon at the A site, (2) the initial binding state (RF compact; pre-accommodated RF-ribosome complex), (3) the open catalytic state (RF open/extended; accommodated RF-ribosome complex) and (4) the state after peptide release. We suggest that the selective advantage of the compact RF-form is that it allows for rapid factor binding into and dissociation from an accuracy maximizing pre-accommodation state.

          Structure-based model. The sequence of states is (1) the termination complex with the stop codon at the A site, (2) the initial binding state (RF compact; “pre-accommodated RF-ribosome complex”), (3) the open catalytic state (RF open/extended; “accommodated RF-ribosome complex”) and (4) the state after peptide release. (The later time point is not known from our experiment, and we only know from another experiment that the final state was seen after 45 s.) Blue: 50S large subunit; orange: 30S small subunit; green: tripeptide; brown: P-tRNA; pink: mRNA; red: compact RF; and blue-purple: extended RF

          Full size image

          Methods

          Components for in vitro translation and fast kinetics

          Buffers and all Escherichia coli (E. coli) components for cell-free protein synthesis were prepared as described22. Ribosomal release complexes (RC) contained tritium (3H) labeled fMet-Phe-Phe-tRNAPhe in the P site and had UAA stop-codon programmed A site. The mRNA sequence used to synthesize the peptide was GGGAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUUAAAUGUUCUUCUAAUGCAGAAAAAAAAAAAAAAAAAAAAA (ORF underlined and bold, SD underlined). Class-1 release factors (RFs), overexpressed in E. coli, had mainly unmethylated glutamine (Q) in the GGQ motif and the RF2 variant contained Ala in position 246. Rate constants for conformational changes of RFs in response to cognate A-site stop codon (kconf) and for ester bond hydrolysis (khydr) at different OH concentrations were estimated as described22. In short, purified release complexes (0.02 µM final concentration) were reacted at 25 °C with saturating amounts of RFs (0.8 µM final) in a quench-flow instrument, and the reaction stopped at different time points by quenching with 17% (final concentration) formic acid. Precipitated [3H]fMet-Phe-Phe-tRNAPhe was separated from the soluble [3H]fMet-Phe-Phe peptide by centrifugation. The amounts of tRNA-bound and free peptides were quantified by scintillation counting of the 3H radiation. Reaction buffer was polymix-HEPES with free Mg2+ concentration adjusted from 5 to 2.5 mM by addition of 2.5 mM Mg2+-chelating UTP. The rate constants for RF association to the A site at 25 °C, ka25, were estimated from their previously published values at 37 °C, ka37 = 60 µM−1 s−1 for RF1 and 23 µM−1 s−1 for RF238 through ka25 = (T2525)·(ŋ37/T37), where T is the absolute temperature and ŋ the water viscosity. Kinetics simulations were carried out with the termination reaction steps modeled as consecutive first-order reactions43.

          Preparation of EM grids and time-resolved cryo-EM

          Quantifoil R1.2/1.3 grids with a 300 mesh size were subjected to glow discharge in H2 and O2 for 25 s using a Solarus 950 plasma cleaning system (Gatan, Pleasanton, CA) set to a power of 10 W. Release complexes and RFs were prepared in the same way as for quench-flow experiments, except the release complexes were unlabeled. For each time point (24 ms and 60 ms), 4 µM of release complexes in polymix-HEPES with 2.5 mM UTP and 6 µM of class-1 release factors in the same buffer were injected into the corresponding microfluidic chip at a rate of 3 µl/s such that they could be mixed and sprayed onto a glow-discharged grid as previously described33. The final concentration of the release complexes and the class-1 release factors after rapid mixing in our microfluidic chip was 2 µM and 3 µM, respectively. As the mixture was sprayed onto the grid, the grid was plunge-frozen in liquid ethane-propane mixture (37%:63%) and stored in liquid nitrogen until it was ready to be imaged.

          Preparation of EM grids and blotting-plunging cryo-EM

          Grids of RC0 and long-incubation complex were prepared with the following protocol. 3 uL sample was applied in the holey grids (gold grids R0.6/1 300 mesh, which was plasma cleaned using the Solarus 950 advanced plasma cleaning system (Gatan, Pleasanton, CA) for 25 s at 10 W using hydrogen and oxygen plasma). Vitrification of samples was performed in a Vitrobot Mark IV (FEI company) at 4 °C and 100% relative humidity by blotting the grids once for 6 s with a blot force 3 before plunging them into the liquid ethane-propane mixture.

          Cryo-EM data collection

          Time-resolved cryo-EM grids were imaged either with a 300 kV Tecnai Polara F30 TEM or a Titan Krios TEM. The images were recorded at a defocus range of −1 to −3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.66 Å or 1.05 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image. Blotting-plunging cryo-EM grids were imaged with a 300 kV Tecnai Polara F30 TEM. The images were recorded at a defocus range of 1–3 µm on a K2 direct detector camera (Gatan, Pleasanton, CA) operating in counting mode with pixel size at 1.24 Å. A total of 40 frames were collected with an electron dose of 8 e/pixel/s for each image.

          Cryo-EM data processing

          The beam-induced motion of the sample and the instability of the stage due to thermal drift was corrected using the MotionCor2 software program44. The contrast transfer function (CTF) of each micrograph was estimated using the CTFFIND4 software program45. Imaged particles were picked using the Autopicker algorithm included in the RELION 2.0 software program46. For each time point (Supplementary Figs 6 and  7), 2D classification of the recorded images were used to separate 70S ribosome-like particles from ice-like and/or debris-like particles picked by the Autopicker algorithm and to classify the particles that were picked for further analysis into 70S ribosome-like particle classes. These particle classes were then combined into a single dataset of 70S ribosome-like particles and subjected to a round of 3D classification for the purpose of eliminating those obvious contaminants from the rest of the dataset. This classification was set for 10 classes with the following sampling parameters: Angular sampling interval of 15°, offset search range of 5 pixels and offset search step of 1 pixel. The sampling parameters were progressively narrowed in the course of the 50 classification iterations, down to 3.7° for the angular sampling interval. At the end of the first classification round, two classes were found inconsistent with the known structure of the 70S ribosomes and were thus rejected. The rest of the particles were regrouped together as one class. All particles from this class were re-extracted using unbinned images. A consensus refinement was calculated using these particles. The A site of the 70S ribosome displays fractioned density indicating heterogeneity, then, therefore, the signal subtraction approach was applied. The A-site density was segmented out of the ribosome using Segger in Chimera47. The mass of density identified as release factor was used for creating a mask in RELION with 3 pixels extension and 6 pixels soft edge using relion_mask_create. This mask was used for subtracting the release factor-like signal from the experimental particles. The new particles images were used directly as input in the masked classification run with the number of particles set for ten classes, and with the mask around the release factor-binding region. This run of focused classification resulted in two separate classes, one with compact and one with extended conformation of the release factors. The corresponding raw particles were finally used to calculated consensus refinements. The local resolution of the final maps was computed using ResMap48.

          For the RC0 complex dataset, the software MotionCor244 was used for motion correction and dose weighting. Gctf49 was used for estimation of the contrast transfer function parameters of each micrograph. RELION46 was used for all other image processing steps. Particles picking was done automatically in RELION. Boxed out particles were extracted from dose-weighted micrographs with eight times binning. 2D classifications were initially performed on bin8 particle stacks to remove false positive particles from the particle picking step. 3D classification were performed on bin4 particle stacks. Classes from bin4 and bin2 3D classification showing high-resolution features were saved for further processing steps. Un-binned particles from this class were re-extracted and subjected to auto-refinement. The final density map was sharpened by applying a negative B-factor estimated by automated procedures. Local resolution variations were estimated using ResMap48 and visualized with UCSF Chimera47.

          Model building and refinement

          Models of the E. coli 70S ribosome (5MDV, 5MDW, and 5DFE) were docked into the maps using UCSF Chimera. The pixel size was calibrated by creating the density map from the atomic model and changing the pixel size of the map to maximize the cross-correlation value. For the compact RF1 model, a homology model was generated with the crystal structure of the RF1 (PDB ID: 1ZBT) as a template using the SWISS-MODEL online server50. This homology model was rigid-body-fitted into the map using UCSF Chimera, followed by manual adjustment in Coot51. Due to the lack of density, domain I of RF1 was not modeled.

          Figure preparation

          All figures showing electron densities and atomic models were generated using UCSF Chimera47.

          Data availability

          The data that support the findings of this study are available from the corresponding author upon request. The atomic coordinates and the associated maps have been deposited in the PDB and EMDB with the accession codes 20173, 20184, 20187, 20188, 20193, 20204, 6ORE, 6ORL, 6OSQ, 6OST, 6OT3, and 6OUO. The source data underlying Supplementary Figs 2 and 3 are provided as a Source Data file.

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          Acknowledgements

          This work was supported by HHMI and grants NIH R01 GM55440 and R01 GM29169 (to J.F.), the Swedish Research Council, and the Knut and Alice Wallenberg Foundation (to M.E.), and the Sederholms travel stipend (Uppsala University) (to G.I.).

          Author information

          Author notes
          1. These authors contributed equally: Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar.

          Affiliations

          1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032, USA

            Ziao Fu, Sandip Kaledhonkar, Robert A. Grassucci & Joachim Frank

          2. Department of Cell and Molecular Biology, Uppsala University, Uppsala, 751 24, Sweden

            Gabriele Indrisiunaite & Måns Ehrenberg

          3. Department of Biological Sciences, Barnard College, New York, NY, 10027, USA

            Binita Shah

          4. Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

            Ming Sun, Bo Chen & Joachim Frank

          Contributions

          Z.F., G.I., S.K., B.S., M.S., B.C., R.A.G., M.E., and J.F. conceived and designed experiments. G.I. carried out biochemical experiments. Z.F., S.K., G.I., and B.S. performed time-resolved cryo-EM experiments. Z.F., S.K., G.I., B.S., and M.S. performed image processing and atomic modeling. Z.F., S.K., G.I., and B.S. analyzed the data. Z.F., G.I., S.K., M.E., and J.F. wrote the manuscript.

          Corresponding author

          Correspondence to Joachim Frank.

          Ethics declarations

          Competing interests

          The authors declare no competing interests.

          Additional information

          Journal peer review information:Nature Communications thanks Andrei Korostelev, Joseph Puglisi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

          Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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          Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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          Источник: https://www.nature.com/articles/s41467-019-10608-z
          , , ¶

          Specifies an address to listen on for a stream (), datagram (), or sequential packet () socket, respectively. The address can be written in various formats:

          If the address starts with a slash (""), it is read as file system socket in the socket family.

          If the address starts with an at symbol (""), it is read as abstract namespace socket in the family. The "" is replaced with a character before binding. For details, see unix(7).

          If the address string is a single number, it is read as port number to listen on via IPv6. Depending on the value of (see below) this might result in the service being available via both IPv6 and IPv4 (default) or just via IPv6.

          If the address string is a string in the format "", it is interpreted as IPv4 address and port .

          If the address string is a string in the format "", it is interpreted as IPv6 address and port . An optional interface scope (interface name or number) may be specified after a "" symbol: "". Interface scopes are only useful with link-local addresses, because the kernel ignores them in other cases. Note that if an address is specified as IPv6, it might still make the service available via IPv4 too, depending on the setting (see below).

          If the address string is a string in the format "", it is read as CID on a port address in the family. The CID is a unique 32-bit integer identifier in analogous to an IP address. Specifying the CID is optional, and may be set to the empty string.

          Note that (i.e. ) is only available for sockets. (i.e. ) when used for IP sockets refers to TCP sockets, (i.e. ) to UDP.

          These options may be specified more than once, in which case incoming traffic on any of the sockets will trigger service activation, and all listed sockets will be passed to the service, regardless of whether there is incoming traffic on them or not. If the empty string is assigned to any of these options, the list of addresses to listen on is reset, all prior uses of any of these options will have no effect.

          It is also possible to have more than one socket unit for the same service when using , and the service will receive all the sockets configured in all the socket units. Sockets configured in one unit are passed in the order of configuration, but no ordering between socket units is specified.

          If an IP address is used here, it is often desirable to listen on it before the interface it is configured on is up and running, and even regardless of whether it will be up and running at any point. To deal with this, it is recommended to set the option described below.

          Specifies a file system FIFO (see fifo(7) for details) to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above.

          Specifies a special file in the file system to listen on. This expects an absolute file system path as argument. Behavior otherwise is very similar to the directive above. Use this to open character device nodes as well as special files in and .

          Specifies a Netlink family to create a socket for to listen on. This expects a short string referring to the family name (such as or ) as argument, optionally suffixed by a whitespace followed by a multicast group integer. Behavior otherwise is very similar to the directive above.

          Specifies a POSIX message queue name to listen on (see mq_overview(7) for details). This expects a valid message queue name (i.e. beginning with ""). Behavior otherwise is very similar to the directive above. On Linux message queue descriptors are actually file descriptors and can be inherited between processes.

          Specifies a USB FunctionFS endpoints location to listen on, for implementation of USB gadget functions. This expects an absolute file system path of a FunctionFS mount point as the argument. Behavior otherwise is very similar to the directive above. Use this to open the FunctionFS endpoint . When using this option, the activated service has to have the and options set.

          Takes one of or . The socket will use the UDP-Lite () or SCTP () protocol, respectively.

          Takes one of , or . Controls the IPV6_V6ONLY socket option (see ipv6(7) for details). If , IPv6 sockets bound will be accessible via both IPv4 and IPv6. If , they will be accessible via IPv6 only. If (which is the default, surprise!), the system wide default setting is used, as controlled by , which in turn defaults to the equivalent of .

          Takes an unsigned integer argument. Specifies the number of connections to queue that have not been accepted yet. This setting matters only for stream and sequential packet sockets. See listen(2) for details. Defaults to SOMAXCONN (128).

          Specifies a network interface name to bind this socket to. If set, traffic will only be accepted from the specified network interfaces. This controls the socket option (see socket(7) for details). If this option is used, an implicit dependency from this socket unit on the network interface device unit is created (see systemd.device(5)). Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

          , ¶

          Takes a UNIX user/group name. When specified, all sockets and FIFO nodes in the file system are owned by the specified user and group. If unset (the default), the nodes are owned by the root user/group (if run in system context) or the invoking user/group (if run in user context). If only a user is specified but no group, then the group is derived from the user's default group.

          If listening on a file system socket or FIFO, this option specifies the file system access mode used when creating the file node. Takes an access mode in octal notation. Defaults to 0666.

          If listening on a file system socket or FIFO, the parent directories are automatically created if needed. This option specifies the file system access mode used when creating these directories. Takes an access mode in octal notation. Defaults to 0755.

          Takes a boolean argument. If yes, a service instance is spawned for each incoming connection and only the connection socket is passed to it. If no, all listening sockets themselves are passed to the started service unit, and only one service unit is spawned for all connections (also see above). This value is ignored for datagram sockets and FIFOs where a single service unit unconditionally handles all incoming traffic. Defaults to . For performance reasons, it is recommended to write new daemons only in a way that is suitable for . A daemon listening on an socket may, but does not need to, call close(2) on the received socket before exiting. However, it must not unlink the socket from a file system. It should not invoke shutdown(2) on sockets it got with , but it may do so for sockets it got with set. Setting is mostly useful to allow daemons designed for usage with inetd(8) to work unmodified with systemd socket activation.

          For IPv4 and IPv6 connections, the environment variable will contain the remote IP address, and will contain the remote port. This is the same as the format used by CGI. For , the port is the IP protocol.

          Takes a boolean argument. May only be used in conjunction with . If true, the specified special file is opened in read-write mode, if false, in read-only mode. Defaults to false.

          Takes a boolean argument. May only be used when . If yes, the socket's buffers are cleared after the triggered service exited. This causes any pending data to be flushed and any pending incoming connections to be rejected. If no, the socket's buffers won't be cleared, permitting the service to handle any pending connections after restart, which is the usually expected behaviour. Defaults to .

          The maximum number of connections to simultaneously run services instances for, when is set. If more concurrent connections are coming in, they will be refused until at least one existing connection is terminated. This setting has no effect on sockets configured with or datagram sockets. Defaults to 64.

          The maximum number of connections for a service per source IP address. This is very similar to the directive above. Disabled by default.

          Takes a boolean argument. If true, the TCP/IP stack will send a keep alive message after 2h (depending on the configuration of ) for all TCP streams accepted on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults to .

          Takes time (in seconds) as argument. The connection needs to remain idle before TCP starts sending keepalive probes. This controls the TCP_KEEPIDLE socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 7200 seconds (2 hours).

          Takes time (in seconds) as argument between individual keepalive probes, if the socket option has been set on this socket. This controls the socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 75 seconds.

          Takes an integer as argument. It is the number of unacknowledged probes to send before considering the connection dead and notifying the application layer. This controls the TCP_KEEPCNT socket option (see socket(7) and the TCP Keepalive HOWTO for details.) Defaults value is 9.

          Takes a boolean argument. TCP Nagle's algorithm works by combining a number of small outgoing messages, and sending them all at once. This controls the TCP_NODELAY socket option (see tcp(7)). Defaults to .

          Takes an integer argument controlling the priority for all traffic sent from this socket. This controls the socket option (see socket(7) for details.).

          Takes time (in seconds) as argument. If set, the listening process will be awakened only when data arrives on the socket, and not immediately when connection is established. When this option is set, the socket option will be used (see tcp(7)), and the kernel will ignore initial ACK packets without any data. The argument specifies the approximate amount of time the kernel should wait for incoming data before falling back to the normal behavior of honoring empty ACK packets. This option is beneficial for protocols where the client sends the data first (e.g. HTTP, in contrast to SMTP), because the server process will not be woken up unnecessarily before it can take any action.

          If the client also uses the option, the latency of the initial connection may be reduced, because the kernel will send data in the final packet establishing the connection (the third packet in the "three-way handshake").

          Disabled by default.

          , ¶

          Takes an integer argument controlling the receive or send buffer sizes of this socket, respectively. This controls the and socket options (see socket(7) for details.). The usual suffixes K, M, G are supported and are understood to the base of 1024.

          Takes an integer argument controlling the IP Type-Of-Service field for packets generated from this socket. This controls the socket option (see ip(7) for details.). Either a numeric string or one of , , or may be specified.

          Takes an integer argument controlling the IPv4 Time-To-Live/IPv6 Hop-Count field for packets generated from this socket. This sets the / socket options (see ip(7) and ipv6(7) for details.)

          Takes an integer value. Controls the firewall mark of packets generated by this socket. This can be used in the firewall logic to filter packets from this socket. This sets the socket option. See iptables(8) for details.

          Takes a boolean value. If true, allows multiple bind(2)s to this TCP or UDP port. This controls the socket option. See socket(7) for details.

          , , ¶

          Takes a string value. Controls the extended attributes "", "" and "", respectively, i.e. the security label of the FIFO, or the security label for the incoming or outgoing connections of the socket, respectively. See Smack.txt for details.

          Takes a boolean argument. When true, systemd will attempt to figure out the SELinux label used for the instantiated service from the information handed by the peer over the network. Note that only the security level is used from the information provided by the peer. Other parts of the resulting SELinux context originate from either the target binary that is effectively triggered by socket unit or from the value of the option. This configuration option applies only when activated service is passed in single socket file descriptor, i.e. service instances that have standard input connected to a socket or services triggered by exactly one socket unit. Also note that this option is useful only when MLS/MCS SELinux policy is deployed. Defaults to "".

          Takes a size in bytes. Controls the pipe buffer size of FIFOs configured in this socket unit. See fcntl(2) for details. The usual suffixes K, M, G are supported and are understood to the base of 1024.

          , ¶

          These two settings take integer values and control the mq_maxmsg field or the mq_msgsize field, respectively, when creating the message queue. Note that either none or both of these variables need to be set. See mq_setattr(3) for details.

          Takes a boolean value. Controls whether the socket can be bound to non-local IP addresses. This is useful to configure sockets listening on specific IP addresses before those IP addresses are successfully configured on a network interface. This sets the / socket option. For robustness reasons it is recommended to use this option whenever you bind a socket to a specific IP address. Defaults to .

          Takes a boolean value. Controls the / socket option. Defaults to .

          Takes a boolean value. This controls the socket option, which allows broadcast datagrams to be sent from this socket. Defaults to .

          Takes a boolean value. This controls the socket option, which allows sockets to receive the credentials of the sending process in an ancillary message. Defaults to .

          Takes a boolean value. This controls the socket option, which allows sockets to receive the security context of the sending process in an ancillary message. Defaults to .

          Takes a boolean value. This controls the , , or socket options, which enable reception of additional per-packet metadata as ancillary message, on , , and sockets. Defaults to .

          Takes one of "", "" (alias: "", "") or "" (alias: ""). This controls the or socket options, and enables whether ingress network traffic shall carry timestamping metadata. Defaults to .

          Takes a string value. Controls the TCP congestion algorithm used by this socket. Should be one of "", "", "", "" or any other available algorithm supported by the IP stack. This setting applies only to stream sockets.

          , ¶

          Takes one or more command lines, which are executed before or after the listening sockets/FIFOs are created and bound, respectively. The first token of the command line must be an absolute filename, then followed by arguments for the process. Multiple command lines may be specified following the same scheme as used for of service unit files.

          , ¶

          Additional commands that are executed before or after the listening sockets/FIFOs are closed and removed, respectively. Multiple command lines may be specified following the same scheme as used for of service unit files.

          Configures the time to wait for the commands specified in , , and to finish. If a command does not exit within the configured time, the socket will be considered failed and be shut down again. All commands still running will be terminated forcibly via , and after another delay of this time with . (See in systemd.kill(5).) Takes a unit-less value in seconds, or a time span value such as "5min 20s". Pass "" to disable the timeout logic. Defaults to from the manager configuration file (see systemd-system.conf(5)).

          Specifies the service unit name to activate on incoming traffic. This setting is only allowed for sockets with . It defaults to the service that bears the same name as the socket (with the suffix replaced). In most cases, it should not be necessary to use this option. Note that setting this parameter might result in additional dependencies to be added to the unit (see above).

          Takes a boolean argument. If enabled, any file nodes created by this socket unit are removed when it is stopped. This applies to sockets in the file system, POSIX message queues, FIFOs, as well as any symlinks to them configured with . Normally, it should not be necessary to use this option, and is not recommended as services might continue to run after the socket unit has been terminated and it should still be possible to communicate with them via their file system node. Defaults to off.

          Takes a list of file system paths. The specified paths will be created as symlinks to the socket path or FIFO path of this socket unit. If this setting is used, only one socket in the file system or one FIFO may be configured for the socket unit. Use this option to manage one or more symlinked alias names for a socket, binding their lifecycle together. Note that if creation of a symlink fails this is not considered fatal for the socket unit, and the socket unit may still start. If an empty string is assigned, the list of paths is reset. Defaults to an empty list.

          Assigns a name to all file descriptors this socket unit encapsulates. This is useful to help activated services identify specific file descriptors, if multiple fds are passed. Services may use the sd_listen_fds_with_names(3) call to acquire the names configured for the received file descriptors. Names may contain any ASCII character, but must exclude control characters and "", and must be at most 255 characters in length. If this setting is not used, the file descriptor name defaults to the name of the socket unit, including its suffix.

          , ¶

          Configures a limit on how often this socket unit my be activated within a specific time interval. The may be used to configure the length of the time interval in the usual time units "", "", "", "", "", … and defaults to 2s (See systemd.time(7) for details on the various time units understood). The setting takes a positive integer value and specifies the number of permitted activations per time interval, and defaults to 200 for sockets (thus by default permitting 200 activations per 2s), and 20 otherwise (20 activations per 2s). Set either to 0 to disable any form of trigger rate limiting. If the limit is hit, the socket unit is placed into a failure mode, and will not be connectible anymore until restarted. Note that this limit is enforced before the service activation is enqueued.

          Источник: https://www.freedesktop.org/software/systemd/man/systemd.socket.html

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