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

eLife ◽  
2016 ◽  
Vol 5 ◽  
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.

scholarly journals Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joshua W. McCausland ◽  
Xinxing Yang ◽  
Georgia R. Squyres ◽  
Zhixin Lyu ◽  
Kevin E. Bruce ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Theoretical Modelling ◽  
Binding Potential ◽  
Central Component ◽  
Macromolecular Complex ◽  
Bacterial Cells ◽  
Brownian Ratchet ◽  
Ratchet Mechanism ◽  
Directional Movement

AbstractThe FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

scholarly journals Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

2021 ◽  
Author(s):  
Steffen Wolf ◽  
Benedikt Sohmen ◽  
Björn Hellenkamp ◽  
Johann Thurn ◽  
Gerhard Stock ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Md Simulations ◽  
Large Protein ◽  
Single Molecule Fret ◽  
Fluorescence Methods ◽  
Dynamics Simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.

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

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.

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

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).

scholarly journals Transport mechanism of a glutamate transporter homologue GltPh

2016 ◽  
Vol 44 (3) ◽  
pp. 898-904 ◽  
Author(s):  
Yurui Ji ◽  
Vincent L.G. Postis ◽  
Yingying Wang ◽  
Mark Bartlam ◽  
Adrian Goldman
Keyword(s):  
Single Molecule ◽  
Transport Mechanism ◽  
Glutamate Transporter ◽  
Md Simulations ◽  
Research Progress ◽  
Pyrococcus Horikoshii ◽  
Single Molecule Fret ◽  
Neurotransmitter Glutamate ◽  
Central Nervous Systems ◽  
Recent Research Progress

Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter.

scholarly journals The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

2019 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Md Simulations ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Single Molecule Fret ◽  
Allosteric Mechanism ◽  
Specific Transport ◽  
Slc6 Family ◽  
The Stability

AbstractNeurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

scholarly journals Local-to-global signal transduction at the core of the Mn2+ sensing riboswitch

2019 ◽  
Author(s):  
Krishna C Suddala ◽  
Ian R Price ◽  
Michal Janeček ◽  
Petra Kührová ◽  
Shiba Dandpat ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Metal Ions ◽  
Single Molecule ◽  
Md Simulations ◽  
Xanthomonas Oryzae ◽  
Manganese Ion ◽  
The Core ◽  
Ion Sensing ◽  
Single Molecule Fret ◽  
Global Signal

The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 μs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

scholarly journals A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Erich Stefan ◽  
Susanne Hofmann ◽  
Robert Tampé
Keyword(s):  
Abc Transporter ◽  
Antigen Processing ◽  
Atp Hydrolysis ◽  
Power Stroke ◽  
Atp Binding ◽  
Nucleotide Exchange ◽  
Single Turnover ◽  
Physiological Processes ◽  
Chemomechanical Coupling ◽  
Functional Homolog

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

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