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.

• Keywords
• Dynamic Allostery, Protein Translocation, SecA, SecYEG, Single‐Molecule FRET,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Adenosine Triphosphatases genetics   metabolism   MeSH
• Bacterial Proteins * metabolism   MeSH
• Nucleotides metabolism   MeSH
• SecA Proteins metabolism   MeSH
• Escherichia coli Proteins * metabolism   MeSH
• SEC Translocation Channels chemistry   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Adenosine Triphosphatases MeSH
• Bacterial Proteins * MeSH
• Nucleotides MeSH
• SecA Proteins MeSH
• Escherichia coli Proteins * MeSH
• SEC Translocation Channels MeSH

The β-barrel assembly machinery (BAM) catalyses the folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The β-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.

• MeSH
• Catalysis MeSH
• Protein Conformation MeSH
• Lipid Bilayers * MeSH
• Lipids * MeSH
• Protein Multimerization * MeSH
• Multiprotein Complexes chemistry   metabolism   MeSH
• Nanostructures * MeSH
• Bacterial Outer Membrane Proteins chemistry   metabolism   MeSH
• Proteolipids metabolism   MeSH
• Protein Folding MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipid Bilayers * MeSH
• Lipids * MeSH
• Multiprotein Complexes MeSH
• Bacterial Outer Membrane Proteins MeSH
• Proteolipids MeSH
• proteoliposomes MeSH Browser

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
• Names of Substances
• Molecular Chaperones MeSH
• Peptidylprolyl Isomerase MeSH
• Bacterial Outer Membrane Proteins MeSH
• Escherichia coli Proteins MeSH
• SurA protein, E coli MeSH Browser
• Carrier Proteins MeSH

Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

• Keywords
• E. coli, SecA, SecYEG, biochemistry, chemical biology, computational biology, electron paramagnetic resonance (EPR) spectroscopy, hydrogen deuterium exchange (HDX) mass spectrometry, molecular dynamics, protein translocation, systems biology,
• MeSH
• Adenosine Triphosphate chemistry   metabolism   MeSH
• Adenosine Triphosphatases chemistry   metabolism   MeSH
• Escherichia coli metabolism   MeSH
• Membrane Transport Proteins chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Protein Precursors metabolism   MeSH
• SecA Proteins chemistry   metabolism   MeSH
• Escherichia coli Proteins chemistry   metabolism   MeSH
• Protein Folding * MeSH
• SEC Translocation Channels chemistry   metabolism   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Adenosine Triphosphatases MeSH
• Membrane Transport Proteins MeSH
• Protein Precursors MeSH
• SecA Proteins MeSH
• Escherichia coli Proteins MeSH
• SEC Translocation Channels MeSH