Dynamic action of the Sec machinery during initiation, protein translocation and termination

Fessl, Tomáš; Watkins, Daniel W.; Oatley, Peter; Allen, William J.; Corey, Robin A.; Horne, Jim E.; Baldwin, Steve; Radford, Sheena E.; Collinson, Ian; Tůma, Roman

doi:10.7554/elife.35112

Cited by 59 publications

(83 citation statements)

References 59 publications

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“…Again, conversely those identified regions would be stabilised after hydrolysis to ADP. These observations are consistent with an ATP-driven opening of the SecY channel, and closure after hydrolysis 7,8 .…”

Section: Mainsupporting

confidence: 87%

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Ahdash

Pyle

Allen

et al. 2019

Preprint

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The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

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Section: Mainsupporting

confidence: 87%

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Ahdash

Pyle

Allen

et al. 2019

Preprint

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“…This strategy ensured that all components were present in each observed complex. In contrast, if two different dyes are placed into the same protein 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.…”

Section: Experimental Designmentioning

confidence: 99%

“…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. The large SecY subunit consists of N-and C-terminal halves and forms an hourglass-shaped pore.…”

Section: Introductionmentioning

confidence: 99%

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Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

et al. 2019

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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.

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“…Functions from the FRETBursts package were used to estimate the background signal as a function of time, identify and remove artefacts due to photophysical effects such as blinking, and provide an optimal signal to noise ratio. To obtain EFRET values three correction parameters were applied as described previously 83 : γ-factor (to account for differences in the efficiency of excitation of each dye), donor leakage into the acceptor channel and acceptor direct excitation by the donor excitation laser. The data from each 10 minute acquisition was merged prior to subsequent analysis.…”

Section: Single Molecule Förster Resonance Energy Transfer (Smfret)mentioning

confidence: 99%

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

Calabrese

Schiffrin

Watson

et al. 2019

Preprint

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AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

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Dynamic action of the Sec machinery during initiation, protein translocation and termination

Cited by 59 publications

References 59 publications

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

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