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

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

eLife ◽

10.7554/elife.47402 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William John Allen ◽

Robin A Corey ◽

Ian Collinson ◽

...

Keyword(s):

Conformational Changes ◽

Opposite Effect ◽

Protein Transport ◽

Protein Translocation ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

The bacterial Sec translocon is a multi-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 pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.

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Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis

Science Advances ◽

10.1126/sciadv.aat8797 ◽

2018 ◽

Vol 4 (10) ◽

pp. eaat8797 ◽

Cited By ~ 8

Author(s):

Nagaraju Chada ◽

Kanokporn Chattrakun ◽

Brendan P. Marsh ◽

Chunfeng Mao ◽

Priya Bariya ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Conformational Dynamics ◽

Real Space ◽

Biochemical Activity ◽

Force Microscopy ◽

Atomic Force ◽

Reversible Transitions ◽

Space Measurement

SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.

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Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

PeerJ ◽

10.7717/peerj.9408 ◽

2020 ◽

Vol 8 ◽

pp. e9408

Author(s):

Shanshan Wu ◽

Tam T.T.N. Nguyen ◽

Olga V. Moroz ◽

Johan P. Turkenburg ◽

Jens E. Nielsen ◽

...

Keyword(s):

Conformational Changes ◽

Solvent Accessibility ◽

Conformational Dynamics ◽

Structural Heterogeneity ◽

Catalytic Process ◽

Conformational Heterogeneity ◽

X Ray ◽

Exchange Mass Spectrometry ◽

Bacillus Lentus ◽

Hdx Ms

Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

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Ultrafast Brownian-ratchet mechanism for protein translocation by a AAA+ machine

10.1101/2020.11.19.384313 ◽

2020 ◽

Author(s):

Hisham Mazal ◽

Marija Iljina ◽

Inbal Riven ◽

Gilad Haran

Keyword(s):

Single Molecule ◽

Protein Translocation ◽

Conformational Dynamics ◽

Brownian Ratchet ◽

Time Dynamics ◽

Single Molecule Fret ◽

Differential Behavior ◽

Amplitude Fluctuations ◽

Translocation Mechanism ◽

Substrate Proteins

AbstractAAA+ ring-shaped machines, such as ClpB and Hsp104, mediate substrate translocation through their central channel by a set of pore loops. Recent structural studies suggested a universal hand-over-hand translocation mechanism, in which pore loops are moving rigidly in tandem with their corresponding subunits. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB and their response to substrate binding, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond timescale, and change their conformation upon interaction with substrate proteins. Pore-loop conformational dynamics are modulated by nucleotides and strongly correlate with disaggregation activity. The differential behavior of the pore loops along the axial channel points to a fast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process.

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

eLife ◽

10.7554/elife.35112 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 26

Author(s):

Tomas Fessl ◽

Daniel Watkins ◽

Peter Oatley ◽

William John Allen ◽

Robin Adam Corey ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Reaction Pathway ◽

Signal Sequence ◽

Slow Phase ◽

Protein Insertion ◽

Membrane Protein Insertion ◽

Two Phases ◽

Dependent Transport

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.

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Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

10.1101/248310 ◽

2018 ◽

Author(s):

Tomas Fessl ◽

Daniel Watkins ◽

Peter Oatley ◽

William J. Allen ◽

Robin A. Corey ◽

...

Keyword(s):

Protein Secretion ◽

Single Molecule ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Signal Sequence ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Protein Insertion ◽

Initiation Phase ◽

Dependent Transport

AbstractProtein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecYMKEG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allenet al.doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

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

eLife ◽

10.7554/elife.41803 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 15

Author(s):

Robin A Corey ◽

Zainab Ahdash ◽

Anokhi Shah ◽

Euan Pyle ◽

William J Allen ◽

...

Keyword(s):

Conformational Changes ◽

Protein Transport ◽

Protein Translocation ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Protein A ◽

Resonance Spectroscopy ◽

Paramagnetic Resonance ◽

Exchange Mass Spectrometry ◽

Directional Diffusion ◽

Protein Transporters

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.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Nature Communications ◽

10.1038/s41467-020-20032-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

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Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

eLife ◽

10.7554/elife.15598 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 59

Author(s):

William John Allen ◽

Robin Adam Corey ◽

Peter Oatley ◽

Richard Barry Sessions ◽

Steve A Baldwin ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Md Simulations ◽

Atp Binding ◽

Nucleotide Exchange ◽

Brownian Ratchet ◽

Ratchet Mechanism ◽

Single Molecule Fret ◽

Directional Movement

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

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