Biology subjects

Okazaki, K.-i.

Publications and source records attributed to Okazaki, K.-i..

9 recordsLinked to original sources

bioRxiv2026

Conformational Dynamics of Na+-Pumping NADH-Quinone Oxidoreductase during Na+ Translocation from AlphaFold-Facilitated Markov State Modeling

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a respiratory chain enzyme found in pathogenic bacteria, including Vibrio cholerae, and is essential for energy metabolism by generating a transmembrane Na+ gradient that drives ATP synthesis and flagellar motility. Because the molecular structure of Na+-NQR is unrelated to the corresponding mitochondrial H-pumping NADH-quinone oxidoreductase (respiratory complex I), it is a promising antibiotic target. Although it has been shown that Na+ pumping is mediated by an alternating-access conformational change in the NqrD/E subunits, coupled to redox switching of a cofactor, the thermodynamics and kinetics of the conformational transition, including the free-energy profile and the rate-limiting steps, remain unclear. Here, we construct redox-state-dependent Markov state models (MSMs) from extensive molecular dynamics (MD) trajectories in the oxidized and reduced states to quantify the conformational free-energy landscapes and primary transition pathway. To accelerate conformational sampling, MD simulations are initiated from diverse NqrD/E conformations generated by AlphaFold. Our analysis clarifies how the NqrD/E conformation is regulated by the redox state and by Na+ binding to achieve Na+ translocation. This study provides a quantitative framework for understanding ion-pumping mechanisms of redox-driven membrane proteins.

biophysics↗

bioRxiv2025

Enhanced sampling of protein conformations in AlphaFold3 with repulsive bias in the diffusion generative model

Conformational changes in proteins are vital to their function yet remain challenging for state-of-the-art artificial intelligence, such as AlphaFold3 (AF3), to predict. It has been observed that AF3 sometimes fails to capture ligand-induced conformational changes, even though it explicitly includes ligand molecules that induce such changes. To address this challenge, we develop an enhanced sampling scheme that leverages the diffusion-based generative model used in AF3 to predict protein structures. Interpreting the diffusion generative model as a stochastic sampling process analogous to molecular dynamics (MD) simulations, we introduce here a repulsive biasing potential between predicted structures to explore wider conformational space. We demonstrate that the developed model, AF3-ReD, successfully predicts multiple conformational states, including ligand-bound conformations of motor and transporter proteins that the original AF3 does not capture. Compared to another strategy based on multiple sequence alignment (MSA), AF3-ReD predicted intermediate conformations that are relatively closer to the stable states. Thus, AF3-ReD provides a promising approach for understanding dynamic conformational changes associated with ligand binding, including those induced by drug molecules.

biophysics↗

bioRxiv2025

Membrane constriction by dynamin through GTP-driven conformational changes from coarse-grained molecular dynamics simulations

Dynamin, a GTPase involved in membrane remodeling processes, plays a pivotal role in membrane fission during endocytosis. Understanding the molecular mechanisms of dynamin assembly and its conformational change triggered by GTP hydrolysis on lipid membranes is crucial for elucidating the intricate details of membrane constriction and fission events. However, the size and complexity of the dynamin assembly prevent us from obtaining comprehensive molecular mechanisms. In this study, we performed coarse-grained molecular dynamics (CG-MD) simulations to elucidate the chemo-mechanical coupling mechanism of dynamin membrane constriction. We set up a CG Martini simulation system that contains dynamin rings on a tubular membrane with realistic molecular structures. Controlling the length of the tubular membrane with a pressure coupling, we simulate membrane constriction through the nucleotide-state-dependent conformational change of dynamin. The simulation results suggest that the conformational change to the GDP state loosens and expands the dynamin rings, resulting in membrane constriction in the protein-uncoated region. This study lays the groundwork for simulating the chemo-mechanical coupling of dynamin-mediated membrane constriction. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/675253v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@3080b3org.highwire.dtl.DTLVardef@519814org.highwire.dtl.DTLVardef@d76b0forg.highwire.dtl.DTLVardef@b5e4ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

The Na+-pumping mechanism driven by redox reactions in the NADH-quinone oxidoreductase from Vibrio cholerae relies on dynamic conformational changes

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na+-pumping across the membrane. Earlier X-ray and cryo-EM structures of Na+-NQR from Vibrio cholerae suggested that the subunits harboring redox cofactors undergo conformational changes during catalytic turnover. However, these proposed rearrangements have not yet been confirmed. Here, we have identified at least five distinct conformational states of Na+-NQR using: mutants that lack specific cofactors, specific inhibitors or low-sodium conditions. Molecular dynamics simulations based on these structural insights indicate that 2Fe-2S reduction in NqrD/E plays a crucial role in triggering Na+ translocation by driving structural rearrangements in the NqrD/E subunits, which subsequently influence NqrC and NqrF positioning. This study provides the first structural insights into the mechanism of Na+ translocation coupled to electron transfer in Na-NQR.

biochemistry↗

bioRxiv2024

Rotation-direction-dependent regulation of ATPase inhibitory factor 1 for mitochondrial ATP synthase from atomistic simulation

ATPase inhibitory factor 1 (IF1) is an endogenous regulatory protein for mitochondrial FoF1-ATP synthase. It blocks the catalysis and rotation of the F1 part by deeply inserting itself into the rotor-stator interface. Recent single-molecule manipulation experiments have elucidated that forcible rotations only in the ATP-synthesis direction eject IF1, rescuing F1 from the IF1-inhibited state. However, the molecular mechanism of the rotation-direction-dependent process at an atomic resolution is still elusive. Here, we have performed all-atom molecular dynamics (MD) simulations of the IF1-bound F1 structure with a torque applied to the rotor {gamma} subunit. In the torque-applying simulations, we first found that the core part of the {gamma} subunit rotated more in response to an external torque in the synthesis direction than in the hydrolysis direction. Further rotations of the {gamma} subunit up to 120{degrees} revealed that the conformational change of the IF1-bound {beta} was only allowed in the synthesis direction. Also, the 120{degrees} rotation in the synthesis direction disrupted its contacts with IF1, destabilizing the short helix of IF1. After additional rotation up to the synthetic 240{degrees} state, the closed-to-open conformational change of the IF1-bound {beta} subunit pulled IF1 outwardly, deforming the long helix of IF1. These stepwise destabilizations of the IF1 helices should be crucial for IF1 ejection. Our simulations also provided insight into the nullification mechanism of the hydrolytic rotation, highlighting the steric clash between F22 of IF1 and the {beta}TP subunit. Finally, we discussed a sufficient proton motive force to rescue FoF1-ATP synthase from the IF1-inhibited state. Significance statementFoF1-ATP synthase is crucial for cellular energy production. However, its reverse reaction as an ATPase can be harmful, particularly in mammalian cells. This study reveals how ATPase inhibitory factor 1 (IF1) controls the catalysis of mitochondrial ATP synthase and provides the atomic-level description of the rotation-direction-dependent regulation using molecular dynamics simulation. ATP-synthetic {gamma} rotation dissociates IF1 from the complex, thereby recovering active ATP synthesis. However, IF1 completely suppresses the {gamma} rotation through multiple mechanisms to prevent wasteful ATP consumption in ATP-hydrolysis mode. Our analysis also reveals that sufficient torque in the synthesis direction is required to rescue FoF1-ATP synthase from the IF1-inhibited state. These findings offer valuable insights for developing inhibitors by leveraging the structural and functional similarities with IF1.

biophysics↗

bioRxiv2023

Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT

Transporter proteins change their conformation to carry their substrate across the cell membrane. The conformational dynamics are vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed an accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully obtained the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward- open conformation. The results were complemented by the highly accurate structure prediction by AlphaFold2. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/564285v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@194bb47org.highwire.dtl.DTLVardef@19371b3org.highwire.dtl.DTLVardef@10dd0d1org.highwire.dtl.DTLVardef@8552a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

Oxalobacter formigenes is an oxalate-degrading bacterium in the gut microbiota that absorbs food-derived oxalate to use this as a carbon and energy source and thereby helps reduce the risk of kidney stone formation of the host animals 1-4. The bacterial oxalate transporter OxlT uptakes oxalate from the gut to bacterial cells and excrete formate as a degradation product, with a strict discrimination from other carboxylates that serve as nutrients 5-7. Nevertheless, the underlying mechanism remains unclear. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two different conformations, occluded and outward-facing states. The oxalate binding site contains two basic residues that form salt bridges with a dicarboxylate substrate while preventing the conformational switch to the occluded state without an acidic substrate, a disallowed state for an antiporter 8, 9. The occluded ligand-binding pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the binding pocket are completely blocked by extensive interdomain hydrophobic and ionic interactions. Nevertheless, a molecular dynamics simulation showed that a flip of a single side chain neighbouring the substrate is sufficient to trigger the gate opening. The OxlT structure indicates the underlying metabolic interactions enabling favourable symbiosis at a molecular level.

biochemistry↗

bioRxiv2019

Dynamic free energy landscape of a linear motor chitinase from single-particle tracking trajectories

The mechanism of biomolecular motors has been elucidated using single-molecule experiments for visualizing motor motion. However, it remains elusive that how changes in the chemical state during the catalytic cycle of motors lead to unidirectional motions. In this study, we use singlemolecule trajectories to estimate an underlying diffusion model with chemical-state-dependent free energy profile. To consider nonequilibrium trajectories driven by the chemical energy consumed by biomolecular motors, we develop a novel framework based on a hidden Markov model, wherein switching among multiple energy profiles occurs reflecting the chemical state changes in motors. The method is tested using simulation trajectories and applied to singlemolecule trajectories of processive chitinase, a linear motor that is driven by the hydrolysis energy of a single chitin chain. The chemical-state-dependent free energy profile underlying the burnt- bridge Brownian ratchet mechanism of processive chitinase is determined. The novel framework allows us to connect the chemical state changes to the unidirectional motion of biomolecular motors.

biophysics↗

bioRxiv2019

Mechanism of electroneutral sodium/proton antiporter from transition-path shooting

Na+/H+ antiporters exchange sodium ions (Na+) and protons (H+) on opposite sides of lipid membranes, using the gradient of one ion to drive the uphill transport of the other. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP in continuous and unbiased molecular dynamics trajectories that cover the entire transport cycle. We overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations by transition-path shooting. In this way, we selectively capture the rare events in which the six-helix-bundle transporter domain spontaneously moves up and down to shuttle protons and ions across the membrane. The simulations reveal two hydrophobic gates above and below the ion-binding sites that open and close in response to the bundle motion. Weakening the outside gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

biophysics↗