ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Corey, Robin A.; Ahdash, Zainab; Shah, Anokhi; Pyle, Euan; Allen, William J.; Fessl, Tomáš; Lovett, Janet E.; Politis, Argyris; Collinson, Ian

doi:10.7554/elife.41803

Cited by 36 publications

(72 citation statements)

References 81 publications

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“…Our results argue against a proposed ratcheting model, in which the two-helix finger makes only small movements relative to the channel and the polypeptide chain is free to slide in the ATP-bound state Corey et al, 2019). The FRET data indicate that the finger makes in fact very large movements, alternating between a withdrawn conformation and one in which it inserts into the channel.…”

Section: Discussioncontrasting

confidence: 64%

“…In a ratcheting model Corey et al, 2019), the finger serves as a sensor for bulky amino acid residues or short a-helical stretches of the substrate. In a ratcheting model Corey et al, 2019), the finger serves as a sensor for bulky amino acid residues or short a-helical stretches of the substrate.…”

Section: Introductionmentioning

confidence: 99%

“…Several models have been proposed to explain SecA function. In a ratcheting model Corey et al, 2019), the finger serves as a sensor for bulky amino acid residues or short a-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.…”

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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Section: Discussioncontrasting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

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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“…1 µs snapshots were taken from simulations of a pre-protein engaged SecA-SecYE complex, modelled based on PDB 5eul 60 ; see ref 18 for details of the modelling. Two preprotein residues near the cytoplasmic face of SecY were then substituted to either lysine, arginine, glutamate or tryptophan using Scwrl4 51 .…”

Section: Steered MDmentioning

confidence: 99%

“…Previously, we and others have proposed models for the ATP-driven component of secretion [16][17][18] but the mechanism of PMF-stimulated transport has remained elusive. One attractive hypothesis is that negatively charged residues (aspartate and glutamate) experience ∆ψ and are pulled across the membrane electrophoretically 19,20 (Fig.…”

Section: Main Text Introductionmentioning

confidence: 99%

Rate-limiting transport of positive charges through the Sec-machinery is integral to the mechanism of protein transport

Allen

Corey

Watkins

et al. 2019

Preprint

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The proton-motive force (PMF) -the electrochemical gradient of protons across energyconserving membranes -powers protein transport in bacteria, mitochondria and chloroplasts.Here, we propose a 'proton ratchet' mechanism for this process. In the Sec system of bacteria, protons are stripped from lysine side chains of the pre-protein at the cytosolic face of the plasma membrane, then replaced on the exterior, aided by the pH component of the PMF (∆pH; acidic outside). This gives the translocating region of pre-protein a net negative charge, promoting electrophoretic diffusion across the membrane driven by membrane-potential (∆ψ; positive outside). For mitochondrial import (through the TIM23 complex) the proton ratchet acts in the opposite direction, with negatively charged residues protonated for passage across the inner membrane into the negative matrix. The proton ratchet is an elegant solution for coupling the PMF to transport, likely to be used by a range of other transporters of charged molecules.

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A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

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Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research.They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High-resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX-MS as it has matured into an essential tool for membrane protein structural biologists. K E Y W O R D Sallosteric coupling, conformational dynamics, GPCRs, hydrogen-deuterium exchange mass spectrometry, integral membrane proteins, transporters Standard: LC: Reverse-phase chromatography on C18 column with prior trapping for desalting. MS: Often Synapt or Xevo-G2 in MS E mode √ Drift-time aligned MS E (HDMS E ) 17 √ LC column with shorter alkyl chain-for example, BEH C4 or C8 13 √ Gradient optimization (8-30%, 8-50%) 15 √ Saw-tooth gradient after peptides elution to prevent carryover 16 Miscellaneous observationsUse of PEG-based detergents (e.g., Triton X-100) complicates peptides identification. Good practice to start with a benchmark condition-for example, locked protein. 16,18,19 Adding TCEP helps but too much (>1 M) reduces spectral quality. 15Note: The tick indicates parameters that have shown improvement compared with the standard conditions.

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ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Cited by 36 publications

References 81 publications

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

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

Rate-limiting transport of positive charges through the Sec-machinery is integral to the mechanism of protein transport

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

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