Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism
2019; Springer Nature; Volume: 38; Issue: 9 Linguagem: Inglês
10.15252/embj.2018101140
ISSN1460-2075
AutoresMarco A. Catipovic, Benedikt Bauer, Joseph J. Loparo, Tom A. Rapoport,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle15 March 2019free access Source DataTransparent process Protein translocation by the SecA ATPase occurs by a power-stroke mechanism Marco A Catipovic Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Benedikt W Bauer Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Joseph J Loparo Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tom A Rapoport Corresponding Author [email protected] orcid.org/0000-0001-9911-4216 Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Marco A Catipovic Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Benedikt W Bauer Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Joseph J Loparo Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tom A Rapoport Corresponding Author [email protected] orcid.org/0000-0001-9911-4216 Department of Cell Biology, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Marco A Catipovic1,2, Benedikt W Bauer1,2,†, Joseph J Loparo3 and Tom A Rapoport *,1,2 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA 2Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA †Present address: Research Institute of Molecular Pharmacology, Vienna, Austria *Corresponding author. Tel: +1 6174320676; E-mail: [email protected] EMBO J (2019)38:e101140https://doi.org/10.15252/embj.2018101140 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides. Synopsis How AAA ATPases harness energy from ATP to promote directional translocation of polypeptides across membranes is poorly understood. Single-molecule FRET reveals that bacterial SecA uses ATP hydrolysis to push a polypeptide segment into the SecY channel, and phosphate release to slide it through the SecA-SecY complex. SecA two-helix finger and clamp domains move upon ATP binding, hydrolysis and phosphate release. ATP binding moves the two-helix finger towards the SecY channel, causing insertion of the first polypeptide segment into the channel. Upon ATP hydrolysis, finger retraction and clamp domain closure around the translocating polypeptide ensure the translocation progress. Clamp opening induced by phosphate release permits passive sliding of the polypeptide in either direction. Introduction Many processes in the cell involve AAA family ATPases that perform mechanical work to remodel or relocate proteins. Examples include hexameric ATPases, such as the p97 ATPase (Cdc48 in yeast), which extracts proteins from membranes or tight complexes, the Clp's and the ATPases of the 26S proteasome, which push polypeptides into a proteolytic chamber, and the NSF protein, which disassembles SNARE complexes involved in membrane fusion (for review, see Zhao et al, 2007; Bodnar & Rapoport, 2017; Ye et al, 2017; Yedidi et al, 2017). Another important member of this ATPase family is SecA, which translocates polypeptides through the plasma membrane in bacteria (for review, see Corey et al, 2016; Rapoport et al, 2017; Cranford-Smith & Huber, 2018). SecA acts a monomer (Or et al, 2005) and uses the energy of ATP hydrolysis to move its substrates through the protein-conducting SecY channel (Economou & Wickner, 1994). How any of these ATPases perform mechanical work is poorly understood. SecA is a multi-domain protein (Fig EV1A and B) with two nucleotide-binding, RecA-like domains (NBD1 and NBD2), which bind the nucleotide at their interface and move relative to one another during the ATP hydrolysis cycle (Hunt et al, 2002). A two-helix finger, consisting of two helices connected by a loop, inserts into the cytoplasmic opening of the SecY channel (Fig EV1A and B; Zimmer & Rapoport, 2009; Li et al, 2016). A conserved Tyr residue within the loop contacts the translocating polypeptide chain (Erlandson et al, 2008a; Bauer et al, 2014), which is positioned above the SecY channel by a clamp formed by rotation of the polypeptide-crosslinking domain (PPXD) toward NBD2 (Fig EV1B and C). Click here to expand this figure. Figure EV1. Structure of the SecA-SecY complex Side view with a translocating polypeptide (black line) modeled into the crystal structure (PDB 3DIN). SecA is in pink, SecY in blue, and SecG and SecE in green. The lateral gate of the SecY channel is in the front. The two-helix finger (THF) of SecA is highlighted in red and a conserved Tyr residue at its tip shown in yellow in stick presentation. View from the cytosol. SecA's domains are shown in different colors, and ATP is shown in its binding site. The red star indicates the position of the translocating polypeptide. Clamp conformational change. The PPXD from the crystal structure of SecA with an open clamp conformation (PDB 1M74) aligned to SecA in the closed conformation. NBD2 is in blue. The closed PPXD is in yellow and the open conformation is in orange. The arrow indicates the PPXD movement. Download figure Download PowerPoint The SecY channel is formed from three polypeptide chains (SecY, SecE, and SecG). The large SecY subunit consists of N- and C-terminal halves and forms an hourglass-shaped pore. The cytoplasmic cavity is empty, while the extracellular cavity is filled with a plug domain. At the constriction in the middle of the membrane is a pore ring of amino acids. During translocation, the plug is displaced (Li et al, 2016; Fessl et al, 2018), and the polypeptide chain moves through the pore ring across the membrane. Several models have been proposed to explain SecA function. In a ratcheting model (Allen et al, 2016; Corey et al, 2019), the finger serves as a sensor for bulky amino acid residues or short α-helical stretches of the substrate. When such a residue is encountered, SecA converts from the ADP-bound to the ATP-bound state and the SecY channel opens, allowing the residue to diffuse through the pore. Following ATP hydrolysis, the channel closes, trapping the bulky residue on the other side of the membrane. In this model, the finger does not move relative to the channel. By contrast, in a power-stroke model (Bauer et al, 2014), ATP binding at the NBDs would cause the two-helix finger to interact with the polypeptide chain and push it into the channel; following ATP hydrolysis, the finger would disengage and allow free diffusion of the chain in either direction. Here, the finger would undergo large movements toward and away from the channel. In one extreme version of this model, the "plunging model", large domains of SecA would reach entirely through membrane to deliver the substrate to the other side (Economou & Wickner, 1994; Banerjee et al, 2017). As proposed, however, the power-stroke models fail to explain how a SecA domain would return to its starting position without erasing the work done during its power stroke. One possibility is that the clamp holds the polypeptide chain when the two-helix finger resets (Zimmer et al, 2008), but this model seems to be in contradiction with the observation that the polypeptide chain can slide back and forth through the SecA-SecY complex (Erlandson et al, 2008b; Bauer et al, 2014). Recent single-molecule experiments support the idea that the clamp of SecA undergoes nucleotide-dependent conformational changes (Chada et al, 2018; Ernst et al, 2018; Vandenberk et al, 2018), but it remains unclear whether they occur during translocation, as the studies were performed in the absence of SecY and translocation substrate. Here, we use single-molecule Fӧrster resonance energy transfer (FRET) experiments to follow conformational changes of SecA during protein translocation. Single-molecule experiments are required because the ATP hydrolysis cycles of all SecA molecules cannot be synchronized in traditional biochemical assays. Our results show that, upon ATP binding to SecA, the two-helix finger undergoes a large conformational change that pushes the polypeptide into the SecY channel. When the finger resets, the clamp tightens around the polypeptide, thus preserving the progress of translocation. Passive sliding of the polypeptide chain occurs after ATP hydrolysis, when the clamp opens. Our results lead to a comprehensive model for SecA function, which may also be applicable to hexameric ATPases. Results Experimental design We used single-molecule FRET in combination with a reconstituted translocation system (Fig 1A). A translocation intermediate was generated, using purified SecA, SecYEG, and substrate. SecA and SecY were labeled with different fluorophores, and the translocation complex was immobilized on a glass surface via the substrate. This strategy ensured that all components were present in each observed complex. In contrast, if two different dyes are placed into the same protein (Allen et al, 2016; Ernst et al, 2018; Fessl et al, 2018; Vandenberk et al, 2018), one cannot exclude that the unlabeled components are missing and that FRET changes are caused by the dissociation or association of the complex, rather than by conformational changes within the complex. It should be noted that attaching complexes to a glass surface via the SecY channel or lipids yielded very few FRET traces, likely because substrate was absent from many complexes. Also, complexes assembled in vivo required ADP•BeFx during purification, which could not be substituted with other nucleotides in the FRET experiments. Figure 1. SecA's two-helix finger makes large movements during the ATP hydrolysis cycle Experimental setup to measure single-molecule FRET in translocation complexes immobilized on a surface. Cy5 and Cy3 fluorophores were introduced into the two-helix finger of SecA (PDB 3DIN; red space filling model; helices highlighted) at position 809 and into SecY (blue) at position 394, respectively. Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve). As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by gray shading. As in (D), but with ATP (257 traces). Source data are available online for this figure. Source Data for Figure 1 [embj2018101140-sup-0003-SDataFig1.zip] Download figure Download PowerPoint In our specific experimental setup, we introduced single cysteines at different positions into cysteine-lacking Escherichia coli SecA and labeled them with the acceptor fluorophore Cyanine 5 (Cy5). The donor fluorophore (Cy3) was attached to a single cysteine introduced at different positions into cysteine-free E. coli SecY. All SecA and SecY mutants retained translocation and ATPase activity after labeling (Appendix Fig S1 and S2). Proteoliposomes were then reconstituted with labeled SecYEG complex and mixed with labeled SecA, ATP, and substrate. The substrate consisted of a fusion of the first 175 amino acids of proOmpA, including the N-terminal signal sequence (SS), a dihydrofolate reductase (DHFR) domain, and a biotinylation tag. The proteoliposomes were then attached to a coverslip by neutravidin, which interacted with both the biotinylated C-terminus of the substrate and biotinylated polyethylene glycol (PEG) molecules at the surface. In the presence of methotrexate, the DHFR domain of the substrate is tightly folded and too large to move through the SecA-SecY complex, therefore preventing complete translocation of the fusion protein (Bauer & Rapoport, 2009). Essentially, all channels were occupied with translocation intermediate (Appendix Fig S3). In the presence of ATP, the substrate is constantly sliding out of the proteoliposomes and is then pushed back into the SecY channel (Bauer et al, 2014). Thus, despite the fact that, on average, the DHFR domain is abutting the channel, the polypeptide chain is undergoing continuous translocation. FRET was monitored in a flow chamber with wide field total internal reflection fluorescence (TIRF) microscopy. As expected from our setup, fluorescent spots were only detected on the surface in the presence of all components (Appendix Fig S4). Alternating excitation of Cy3 and Cy5 allowed for measurement of both FRET between SecY and SecA, as well as direct detection of SecA. Our experimental design ensured that both partners are present and allowed single SecA molecules to be monitored through many hydrolysis cycles over a period as long as 30 s, i.e., observation times far longer than those allowed by solution FRET experiments. Although the time resolution was limited to 33 ms per frame, this is about 20 times faster than the duration of an ATP hydrolysis cycle measured in bulk (Appendix Fig S2). FRET traces were either obtained in the presence of ATP or the nucleotide was exchanged in the flow chamber to either ADP•BeFx, which mimicks the transition state of ATP hydrolysis, or ATPγS, a slowly hydrolyzing ATP analog. Complexes could not be imaged in the presence of ADP alone, as SecA binds only weakly to SecY in the presence of this nucleotide (Bauer et al, 2014). While the fluorescent spots were stable with nucleotide analogs, they rapidly disappeared in the presence of ATP, likely because SecA dissociates in its ADP-bound state, allowing the substrate to slide backwards in the SecY pore until the entire proteoliposome is released from the glass surface. We therefore added unlabeled SecA when imaging with ATP, keeping the total concentration below the Kd of SecA dimerization (Woodbury et al, 2002) to ensure that only active SecA monomers are observed. The increased concentration allowed SecA to rebind abandoned complexes before they dissociated. Although most rebinding SecA molecules were unlabeled, some were labeled and allowed the observation of FRET over extended time periods. Movement of the two-helix finger of SecA during protein translocation We first analyzed movements of the two-helix finger of SecA. To this end, the donor fluorophore was placed into a periplasmic loop of E. coli SecY (position 394) and the acceptor fluorophore into position 809 of the two-helix finger (Fig 1B). The probes are predicted to be about 50 Å apart according to crystal structures obtained in the presence of the transition state analog ADP•BeFx (Zimmer et al, 2008; Li et al, 2016). Consistent with the observation that ADP•BeFx allows stable binding of SecA to the SecY channel, a static FRET signal between SecA and SecY was observed in all traces (Fig 1C; top trace). Direct excitation of the SecA-bound fluorophore showed that SecA remained bound to the channel (bottom trace). At the end of a trace, the acceptor fluorescence bleached in one step, and the donor fluorescence was de-quenched (see arrow), as expected for a FRET signal. In no case did acceptor fluorescence return, confirming that, in the presence of ADP•BeFx, SecY-bound SecA is not exchanged with SecA in bulk solution. The analysis of many traces showed that the FRET ratios had a Gaussian distribution with a mean value of 0.60 ± 0.12 (Fig 1D). While distance estimates based on FRET probes in a proteinaceous environment are unreliable due to orientation restrictions of the fluorophores, a naïve estimate using the standard FRET equation gives a distance of 51 Å, in close agreement with the structural prediction. In the presence of ATP, SecA repeatedly bound and dissociated from the SecY channel, as demonstrated by direct excitation of the acceptor fluorophore (Fig 1E; bottom trace). While bleaching and dissociation cannot be distinguished a priori in individual traces, the imaging lifetime of individual SecA molecules in the presence of ATP was generally shorter than in the presence of ADP•BeFx (Appendix Fig S5), suggesting that SecA does indeed dissociate in these traces. The FRET signal was highly dynamic when SecA was bound (top trace), alternating between high and low states with mean FRET ratios of 0.90 ± 0.09 and 0.11 ± 0.08 (Fig 1F; additional examples of traces are shown in Appendix Fig S6). The high and low FRET states likely correspond to states in which the two-helix finger is either inserted into or withdrawn from the SecY channel. The large FRET difference indicates that the finger undergoes a substantial conformational change, although its precise movement cannot be deduced from the FRET values. The low FRET state shows a significantly higher occupancy than the high FRET state (Fig 1F). When the donor fluorophore was placed at a different position in SecY (position 103), a markedly similar behavior was observed (Fig EV2A–C). Again, a constant FRET level was observed in the presence of ADP•BeFx, which matched well the estimated inter-fluorophore distance derived from the crystal structures. As before, in the presence of ATP, the FRET traces were dynamic during SecA-bound periods (Fig EV2D). Histograms derived from these traces also showed two populations at low and high FRET levels, with a higher occupancy in the low FRET state (Fig EV2E). Click here to expand this figure. Figure EV2. SecA's two-helix finger movements observed by alternative fluorophore positions Cy5 and Cy3 fluorophores were introduced into the two-helix finger of SecA (PDB 3dIN; red space filling model; helices highlighted) at position 809 and into SecY (blue) at position 103, respectively. Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 106 traces as in (B), fit with a Gaussian model (black curve). As in (B), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by gray shading. As in (C), but with ATP (200 traces). Traces obtained in the presence of different nucleotides were used to determine the number of states best fit by the Markov model. Transition density plot of idealized ATP FRET states obtained in (F). The distribution of dwell times of the low FRET states observed in ATP was fit with exponentials (1,172 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (H), but with high FRET (1,349 high FRET states). Source data are available online for this figure. Download figure Download PowerPoint To connect these FRET changes to the ATP hydrolysis cycle of SecA, we fit the FRET traces with a hidden Markov model (McKinney et al, 2006; Bronson et al, 2009; van de Meent et al, 2014; Fig 2A). These models employ a maximum evidence approach to find the most likely number of structural conformations that underlie the observed data. Individual traces were fit with an increasing complexity of models, which were scored positively for the closeness of their fit to the data and negatively for the number of discrete FRET states included. In this way, the most parsimonious model was selected that reproduces the data without evoking extraneous conformations (Bronson et al, 2009). This analysis confirmed that in the presence of ADP•BeFx only one conformational state exists, while in the presence of ATP, and with both donor positions, the majority of traces showed two states (Figs 2B and EV2F). Transitions between these idealized FRET states can also be plotted as transition density plots (TDPs) to show how these FRET states connect to each other (McKinney et al, 2006). Transition density plots of idealized FRET states obtained in the presence of ATP showed symmetry across the principal diagonal, indicating cycling between only two FRET states (Figs 2C and EV2G). Thus, the high and low FRET states simply interchange with each other. The distribution of dwell times for the two FRET states observed with ATP could each be fit with a single exponential and demonstrated that the mean lifetime for the low FRET state is about twice as long as that of the high FRET state (Figs 2D and E, and EV2H and I). The low and high FRET states likely correspond to ADP- and ATP-bound states, respectively, as previous experiments showed that SecA spends most of its time during the ATP hydrolysis cycle in the ADP-bound state (Robson et al, 2009). This assumption is consistent with the relatively high intermediate FRET signal observed with the transition state mimic ADP•BeFx (Fig 1C and D). Furthermore, the sum of the high and low FRET lifetimes gives an estimate of the overall ATP hydrolysis rate that agrees with bulk measurements performed at the same temperature (Appendix Fig S2). Finally, FRET experiments with ATPγS, a slowly hydrolyzing analog, also showed two conformational states (Fig 2B), but the high FRET state now lasted as long as the low FRET state (Fig 2F and G). Interestingly, the high FRET value was close to that measured in the presence of ADP•BeFx (0.67 ± 0.11 vs. 0.6 ± 0.11), suggesting that ATPγS extends the duration of the transition state of ATP hydrolysis. Figure 2. The two-helix finger switches between two states A representative FRET trace (blue line) was obtained as in Fig 1 and fit with a hidden Markov model (black dashed line). Traces as in (A) obtained in the presence of different nucleotides were used to determine the number of states best fit by the Markov model. Transition density plot of idealized ATP FRET states obtained in (B). The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1,500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. As in (D), but with high FRET (1,656 high FRET states). Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve). Source data are available online for this figure. Source Data for Figure 2 [embj2018101140-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Given that the average FRET efficiency observed for the two-helix finger is different in the transition state of ATP hydrolysis (ADP•BeFx) and the ADP-bound state (0.6 vs. 0.1), we asked whether the transition between them happens before or after Pi release. We therefore measured FRET in the presence of ADP and Pi, as well as ADP and vanadate (Vi), a phosphate analog that binds more stably. In both conditions, the two-helix finger was primarily in the low FRET state (Fig EV3A–D), indicating that it withdraws before Pi release. The two-helix finger was more dynamic in the presence of ADP•Pi and ADP•Vi than with ADP•BeFx, either because of increased conformational flexibility or frequent dissociation of Pi/Vi. Click here to expand this figure. Figure EV3. Representative FRET data for SecA conformations before Pi release Representative traces for the two-helix finger (positions 809 in SecA and 394 in SecY) obtained with ADP•Pi. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. Distribution of FRET values determined from 227 traces as in (A) fit with a Gaussian model (black curve). As in (A), but with ADP•Vi. As in (B), but with ADP•Vi (202 traces). As in (A), but for the clamp (positions 233 in SecA and 103 in SecY). As in (B), but for the clamp (274 traces). As in (A), but for the clamp with ADP•Vi. As in (B), but for the clamp with ADP•Vi (157 traces). Source data are available online for this figure. Download figure Download PowerPoint Taken together, these results show that, during protein translocation, the two-helix finger of SecA undergoes a large conformational change. It alternates between two conformations during ATP hydrolysis: In the short-lived ATP-bound state, the finger inserts deeply into the SecY channel and gives a high FRET signal, and in the longer ADP-bound state, it withdraws from the pore and produces low FRET. In the transition state, mimicked by ADP•BeFx, the finger is in an intermediate position, but it retracts completely following completion of ATP hydrolysis, before Pi release. Movement of the two-helix finger into the channel would push the polypeptide forward, and movement away would reset the finger for the next cycle. Movement of the clamp of SecA The observation of only two states of the two-helix finger during ATP hydrolysis requires a mechanism that prevents the finger from dragging the polypeptide backwards when the finger moves away from the channel. A likely candidate for holding the polypeptide during finger resetting is the clamp, a groove formed by the rotation of the PPXD toward NBD2 (Fig EV1B and C; Zimmer et al, 2008). Rotation of the PPXD can be inferred from crystal structures of soluble SecA that show this domain at different distances from NBD2 (Hunt et al, 2002; Osborne et al, 2004; Chen et al, 2015), and movement of the translocating polypeptide chain through the clamp is indicated by crosslinking experiments (Bauer & Rapoport, 2009). However, it remained unclear whether the clamp simply forms a conduit for the translocating polypeptide chain or cyclically binds and releases it during ATP hydrolysis. To test whether the clamp undergoes nucleotide-dependent movements, we placed the acceptor fluorophore into the PPXD (position 233) and the donor fluorophore at position 103 in the N-terminal half of SecY (Fig 3A and B). In the presence of ADP•BeFx, a static FRET signal of 0.60 ± 0.08 was observed (Fig 3C and D). Again, the distance estimated with the standard FRET equation agreed well with those measured in crystal structures (Zimmer et al, 2008; Li et al, 2016). In the presence of ATP, we once again observed exchange of SecA molecules on the SecY channel, and changes between two conformations when SecA was bound to the channel (Figs 3E and F, and 4A and B; additional examples of traces are shown in Appendix Fig S7). Similar results were obtained when the donor fluorophore was moved to position 336 in the C-terminal half of SecY (Fig EV4A–H), demonstrating that FRET changes are due to conformational changes of the SecA clamp, rather than the channel. Experiments with the slowly hydrolyzing ATP analog ATPγS showed an increase in the occupancy of the high FRET state (Fig 4C and D), consistent with the clamp movements being linked to ATP hydrolysis. The predominance of the high FRET state in ATPγS indicates that clamp is closed during ATP hydrolysis, though it is unclear whether the initial closure occurs during ATP binding or hydrolysis. Figure 3. SecA's clamp opens and closes during the ATPase cycle Cy5 and Cy3 fluorophores were introduced into the clamp of SecA (PDB 3DIN; red space filling model) at position 233 and into the N-terminal half of SecY (blue) at position 103, respectively. The PPXD and NBD2 making up the clamp are shown as a violet and magenta ribbon models
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