Akio Kitao

Multidimensional replica-exchange method for free-energy calculations

We have developed a new simulation algorithm for free-energy calculations. The method is a multidimensional extension of the replica-exchange method. While pairs of replicas with different temperatures are exchanged during the simulation in the original replica-exchange method, pairs of replicas with different temperatures and/or different parameters of the potential energy are exchanged in the new algorithm. This greatly enhances the sampling of the conformational space and allows accurate calculations of free energy in a wide temperature range from a single simulation run, using the weighted histogram analysis method.

Investigating protein dynamics in collective coordinate space.

Currently, collective coordinates are commonly employed in order to examine protein dynamics. In recent studies, they have been successfully applied to finding functionally relevant motions, to investigating the physical nature of protein dynamics, to sampling of the conformational space and to the analysis of experimental data. Collective coordinates also have other possible applications.

Model-Free Methods of Analyzing Domain Motions in Proteins from Simulation: A Comparison of Normal Mode Analysis and Molecular Dynamics Simulation of Lysozyme

Model‐free methods are introduced to determine quantities pertaining to protein domain motions from normal mode analyses and molecular dynamics simulations. For the normal mode analysis, the methods are based on the assumption that in low frequency modes, domain motions can be well approximated by modes of motion external to the domains. To analyze the molecular dynamics trajectory, a principal component analysis tailored specifically to analyze interdomain motions is applied. A method based on the curl of the atomic displacements is described, which yields a sharp discrimination of domains, and which defines a unique interdomain screw‐axis. Hinge axes are defined and classified as twist or closure axes depending on their direction. The methods have been tested on lysozyme. A remarkable correspondence was found between the first normal mode axis and the first principal mode axis, with both axes passing within 3 Å of the alpha‐carbon atoms of residues 2, 39, and 56 of human lysozyme, and near the interdomain helix. The axes of the first modes are overwhelmingly closure axes. A lesser degree of correspondence is found for the second modes, but in both cases they are more twist axes than closure axes. Both analyses reveal that the interdomain connections allow only these two degrees of freedom, one more than provided by a pure mechanical hinge. Proteins 27:425–437, 1997.

Energy landscape of a native protein: Jumping‐among‐minima model

We have investigated energy landscape of human lysozyme in its native state by using principal component analysis and a model, jumping‐among‐minima (JAM) model. These analyses are applied to 1 nsec molecular dynamics trajectory of the protein in water. An assumption embodied in the JAM model allows us to divide protein motions into intra‐substate and inter‐substate motions. By examining intra‐substate motions, it is shown that energy surfaces of individual conformational substates are nearly harmonic and mutually similar. As a result of principal component analysis and JAM model analysis, protein motions are shown to consist of three types of collective modes, multiply hierarchical modes, singly hierarchical modes, and harmonic modes. Multiply hierarchical modes, the number of which accounts only for 0.5% of all modes, dominate contributions to total mean‐square atomic fluctuation. Inter‐substate motions are observed only in a small‐dimensional subspace spanned by the axes of multiplyhierarchical and singly hierarchical modes. Inter‐substate motions have two notable time components: faster component seen within 200 psec and slower component. The former involves transitions among the conformational substates of the low‐level hierarchy, whereas the latter involves transitions of the higher level substates observed along the first four multiply hierarchical modes. We also discuss dependence of the subspace, which contains conformational substates, on time duration of simulation. Proteins 33:496–517, 1998.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Picosecond fluctuating protein energy landscape mapped by pressure–temperature molecular dynamics simulation

Microscopic statistical pressure fluctuations can, in principle, lead to corresponding fluctuations in the shape of a protein energy landscape. To examine this, nanosecond molecular dynamics simulations of lysozyme are performed covering a range of temperatures and pressures. The well known dynamical transition with temperature is found to be pressure-independent, indicating that the effective energy barriers separating conformational substates are not significantly influenced by pressure. In contrast, vibrations within substates stiffen with pressure, due to increased curvature of the local harmonic potential in which the atoms vibrate. The application of pressure is also shown to selectively increase the damping of the anharmonic, low-frequency collective modes in the protein, leaving the more local modes relatively unaffected. The critical damping frequency, i.e., the frequency at which energy is most efficiently dissipated, increases linearly with pressure. The results suggest that an invariant description of protein energy landscapes should be subsumed by a fluctuating picture and that this may have repercussions in, for example, mechanisms of energy dissipation accompanying functional, structural, and chemical relaxation.

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export.

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N‐terminal transmembrane domain (FlhATM) and a C‐terminal cytoplasmic domain (FlhAC). FlhAC is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhAC at 2.8 Å resolution. FlhAC consists of four subdomains (ACD1, ACD2, ACD3 and ACD4) and a linker connecting FlhAC to FlhATM. The sites of temperature‐sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhAC changes its conformation for its function. Molecular dynamics simulation demonstrated an open‐close motion with a 5–10 ns oscillation in the distance between ACD2 and ACD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhAC is essential for protein export.

Hydration Affects Both Harmonic and Anharmonic Nature of Protein Dynamics

To understand the effect of hydration on protein dynamics, inelastic neutron-scattering experiments were performed on staphylococcal nuclease samples at differing hydration levels: dehydrated, partially hydrated, and hydrated. At cryogenic temperatures, hydration affected the collective motions with energies lower than 5 meV, whereas the high-energy localized motions were independent of hydration. The prominent change was a shift of boson peak toward higher energy by hydration, suggesting a hardening of harmonic potential at local minima on the energy landscape. The 240 K transition was observed only for the hydrated protein. Significant quasielastic scattering at 300 K was observed only for the hydrated sample, indicating that the origin of the transition is the motion activated by hydration water. The neutron-scattering profile of the partially hydrated sample was quite similar to that of the hydrated sample at 100 K and 200 K, whereas it was close to the dehydrated sample at 300 K, indicating that partial hydration is sufficient to affect the harmonic nature of protein dynamics, and that there is a threshold hydration level to activate anharmonic motions. Thus, hydration water controls both harmonic and anharmonic protein dynamics by differing means.

Improved protein free energy calculation by more accurate treatment of nonbonded energy: Application to chymotrypsin inhibitor 2, V57A

We developed a software package for improved free energy calculation, in which spherical solvent boundary potential, cell multipole method, and Nosé‐Hoover equation are employed. The performance of the developed software package is demonstrated in the case of valine to alanine mutation of the 57th residue in chymotrypsin inhibitor 2. By using this package, we obtained results quantitatively comparable to experimental results. By the free energy component analysis, it is shown that leucine 51, arginine 65, arginine 67, and phenylalanine 69 residues contribute significantly to the total free energy shift, ΔΔG. Among them, contribution from the hydrophilic arginine 67 residue, which is in close contact with the mutation site, is the largest. Structure around the mutation site is largely changed by the mutation. The structure change is caused mainly by two effects, hydrophobic interaction and short‐range interaction along the sequence. Effects of Nosé‐Hoover algorithm and Kirkwood reaction field are also discussed. Proteins 30:388–400, 1998.

TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P2

Mutations in the ankyrin repeat domain (ARD) of TRPV4 are responsible for several channelopathies, including Charcot-Marie-Tooth disease type 2C and congenital distal and scapuloperoneal spinal muscular atrophy. However, the molecular pathogenesis mediated by these mutations remains elusive, mainly due to limited understanding of the TRPV4 ARD function. Here we show that phosphoinositide binding to the TRPV4 ARD leads to suppression of the channel activity. Among the phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) most potently binds to the TRPV4 ARD. The crystal structure of the TRPV4 ARD in complex with inositol-1,4,5-trisphosphate, the head-group of PI(4,5)P2, and the molecular-dynamics simulations revealed the PI(4,5)P2-binding amino-acid residues. The TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2. Notably, disease-associated TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity. These findings identify TRPV4 ARD as a lipid-binding domain in which interactions with PI(4,5)P2 normalize the channel activity in TRPV4.