The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy genetika metabolismus MeSH
- bakteriální proteiny * metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny SecA metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- translokační kanály SEC chemie MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The 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 its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.
- MeSH
- Escherichia coli metabolismus MeSH
- hmotnostní spektrometrie MeSH
- molekulární chaperony genetika metabolismus MeSH
- peptidylprolylisomerasa genetika metabolismus MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fluorescenční mikroskopie metody MeSH
- hydrolýza MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- mutace MeSH
- proteiny - lokalizační signály genetika MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- protonmotorická síla * MeSH
- translokační kanály SEC chemie genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH