Yasutaka Nishihara

Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

Significance The bacterial flagellum is a molecular machine for the locomotion of bacteria. The flagellar motor can convert the motive force of protons and other ions to molecular motor rotations. We report on the multi-ion transfer mechanism of the flagellar motor stator MotA/B in Escherichia coli, which primarily utilizes protons. We have identified a leucine amino acid residue as the gateway for H3O+ diffusion. Mutations of a key valine residue cause a narrowing of the channel that is consistent with the size-dependent ion selectivity. We also show that the protonation/deprotonation cycle induces a ratchet motion in the stator, which can couple to the motor rotation. Ion selectivity and the ratchet motion are key to understanding the mechanism of the flagellar motor. The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O+ permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor.

Protein Collective Motions Coupled to Ligand Migration in Myoglobin

Ligand migration processes inside myoglobin and protein dynamics coupled to the migration were theoretically investigated with molecular dynamics simulations. Based on a linear response theory, we identified protein motions coupled to the transient migration of ligand, carbon monoxide (CO), through channels. The result indicates that the coupled protein motions involve collective motions extended over the entire protein correlated with local gating motions at the channels. Protein motions, coupled to opening of a channel from the distal pocket to a neighboring xenon site, were found to share the collective motion with experimentally observed protein motions coupled to a doming motion of the heme Fe atom upon photodissociation of the ligand. Analysis based on generalized Langevin dynamics elucidated slow and diffusive features of the protein response motions. Remarkably small transmission coefficients for rates of the CO migrations through myoglobin were found, suggesting that the CO migration dynamics are characterized as motions governed by the protein dynamics involving the collective motions, rather than as thermally activated transitions across energy barriers of well-structured channels.

Parallel cascade selection molecular dynamics for efficient conformational sampling and free energy calculation of proteins

Parallel Cascade Selection Molecular Dynamics (PaCS-MD) was proposed as an efficient conformational sampling method to investigate conformational transition pathway of proteins. In PaCS-MD, cycles of (i) selection of initial structures for multiple independent MD simulations and (ii) conformational sampling by independent MD simulations are repeated until the convergence of the sampling. The selection is conducted so that protein conformation gradually approaches a target. The selection of snapshots is a key to enhance conformational changes by increasing the probability of rare event occurrence. Since the procedure of PaCS-MD is simple, no modification of MD programs is required; the selections of initial structures and the restart of the next cycle in the MD simulations can be handled with relatively simple scripts with straightforward implementation. Trajectories generated by PaCS-MD were further analyzed by the Markov state model (MSM), which enables calculation of free energy landscape. The combinati...

Conformational transition pathway and free energy analyses of proteins by parallel cascade selection molecular dynamics (PaCS-MD)

Parallel Cascade Selection Molecular Dynamics (PaCS-MD) was recently proposed to generate conformational transition pathways of proteins under the condition that a certain target quantity to be reached is introduced (R. Harada and A. Kitao, J. Chem. Phys., 139, 035103 2013). In PaCS-MD, the cycle of short multiple independent molecular dynamics simulations and selection of the structures close to the target quantity for the next cycle are repeated until the simulated structures move sufficiently close to the target. Conformational sampling efficiency is demonstrated in the cases of mini-protein folding/unfolding and protein large conformational transitions. The result of PaCS-MD was further utilized to calculate free energy landscape by the combination with weighted-histogram analysis method or Markov state model.

Structure of the MotA/B Proton Channel

Flagellar motors utilize the motive force of protons and other ions as an energy source. To elucidate the mechanisms of ion permeation and torque generation, it is essential to investigate the structure of the motor stator complex; however, the atomic structure of the transmembrane region of the stator has not been determined experimentally. We recently constructed an atomic model structure of the transmembrane region of the Escherichia coli MotA/B stator complex based on previously published disulfide cross-linking and tryptophan scanning mutations. Dynamic permeation by hydronium ions, sodium ions, and water molecules was then observed using steered molecular dynamics simulations, and free energy profiles for ion/water permeation were calculated using umbrella sampling. We also examined the possible ratchet motion of the cytoplasmic domain induced by the protonation/deprotonation cycle of the MotB proton binding site, Asp32. In this chapter, we describe the methods used to conduct these analyses, including atomic structure modeling of the transmembrane region of the MotA/B complex; molecular dynamics simulations in equilibrium and in ion permeation processes; and ion permeation-free energy profile calculations.