Asymptotics of a Brownian ratchet for protein translocation

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

10.1101/595553 ◽

2019 ◽

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William J. Allen ◽

Robin A. Corey ◽

Ian Collinson ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Protein Transport ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Conformational Dynamics ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

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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A Brownian ratchet for protein translocation including dissociation of ratcheting sites

Journal of Mathematical Biology ◽

10.1007/s00285-012-0519-8 ◽

2012 ◽

Vol 66 (3) ◽

pp. 505-534 ◽

Cited By ~ 3

Author(s):

A. Depperschmidt ◽

N. Ketterer ◽

P. Pfaffelhuber

Keyword(s):

Protein Translocation ◽

Brownian Ratchet

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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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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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