High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration.

High pressure below 100 MPa interferes inter-molecular interactions without causing pressure denaturation of proteins. In Escherichia coli, the binding of the chemotaxis signaling protein CheY to the flagellar motor protein FliM induces reversal of the motor rotation. Using molecular dynamics (MD) simulations and parallel cascade selection MD (PaCS-MD), we show that high pressure increases the water density in the first hydration shell of CheY and considerably induces water penetration into the CheY-FliM interface. PaCS-MD enabled us to observe pressure-induced dissociation of the CheY-FliM complex at atomic resolution. Pressure dependence of binding free energy indicates that the increase of pressure from 0.1 to 100 MPa significantly weakens the binding. Using high-pressure microscopy, we observed that high hydrostatic pressure fixes the motor rotation to the counter-clockwise direction. In conclusion, the application of pressure enhances hydration of the proteins and weakens the binding of CheY to FliM, preventing reversal of the flagellar motor.

Essential ion binding residues for Na+ flow in stator complex of the Vibrio flagellar motor.

The bacterial flagellar motor is a unique supramolecular complex which converts ion flow into rotational force. Many biological devices mainly use two types of ions, proton and sodium ion. This is probably because of the fact that life originated in seawater, which is rich in protons and sodium ions. The polar flagellar motor in Vibrio is coupled with sodium ion and the energy converting unit of the motor is composed of two membrane proteins, PomA and PomB. It has been shown that the ion binding residue essential for ion transduction is the conserved aspartic acid residue (PomB-D24) in the PomB transmembrane region. To reveal the mechanism of ion selectivity, we identified essential residues, PomA-T158 and PomA-T186, other than PomB-D24, in the Na+-driven flagellar motor. It has been shown that the side chain of threonine contacts Na+ in Na+-coupled transporters. We monitored the Na+-binding specific structural changes using ATR-FTIR spectroscopy. The signals were abolished in PomA-T158A and -T186A, as well as in PomB-D24N. Molecular dynamics simulations further confirmed the strong binding of Na+ to D24 and showed that T158A and T186A hindered the Na+ binding and transportation. The data indicate that two threonine residues (PomA-T158 and PomA-T186), together with PomB-D24, are important for Na+ conduction in the Vibrio flagellar motor. The results contribute to clarify the mechanism of ion recognition and conversion of ion flow into mechanical force.

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.

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

The escape process of carbon monoxide from myoglobin to solution at physiological temperature.

The carbon monoxide (CO) docking sites involved in the ligand escape process from the iron atom in hem of myoglobin (Mb) to solution at physiological temperature were studied on the basis of the effect of xenon (Xe) on the ligand escape rate by the transient grating (TG) technique. The TG method provides a direct measurement of the changes in molecular volume. The apparent CO escaping rate and the volume contraction increase with increasing Xe pressure. The pressure dependence of the rate is consistent with that of the Xe population at the Xe(1) site. This result clearly shows that CO is trapped at the Xe(1) site before escaping to solvent in a Xe-free solution at room temperature. It is shown that only CO but not the trapped Xe is released by the photoexcitation of the Xe-trapped MbCO. A dissociation scheme is proposed to explain the enhancement of the escaping rate by the presence of Xe(1). There are two branches for the CO escaping pathway. The dominant part of the dissociated CO escapes to the solvent through the Xe(1) trapping site under the Xe-free condition, and there are at least three intermediate states along this pathway. When a Xe atom blocks the Xe(1) site, the CO escapes through another route.