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 metabolism   MeSH
• Mass Spectrometry MeSH
• Molecular Chaperones genetics   metabolism   MeSH
• Peptidylprolyl Isomerase genetics   metabolism   MeSH
• Bacterial Outer Membrane Proteins genetics   metabolism   MeSH
• Escherichia coli Proteins genetics   metabolism   MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Adenosine Triphosphate metabolism   MeSH
• Adenosine Triphosphatases chemistry   genetics   metabolism   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Microscopy, Fluorescence methods   MeSH
• Hydrolysis MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Protein Sorting Signals genetics   MeSH
• Escherichia coli Proteins chemistry   genetics   metabolism   MeSH
• Proton-Motive Force * MeSH
• SEC Translocation Channels chemistry   genetics   metabolism   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH