Biology subjects

Ohnuki, J.

Publications and source records attributed to Ohnuki, J..

3 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↗

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↗