Kazuhiro Takemura

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.

Evaluation of protein-protein docking model structures using all-atom molecular dynamics simulations combined with the solution theory in the energy representation.

We propose a method to evaluate binding free energy differences among distinct protein-protein complex model structures through all-atom molecular dynamics simulations in explicit water using the solution theory in the energy representation. Complex model structures are generated from a pair of monomeric structures using the rigid-body docking program ZDOCK. After structure refinement by side chain optimization and all-atom molecular dynamics simulations in explicit water, complex models are evaluated based on the sum of their conformational and solvation free energies, the latter calculated from the energy distribution functions obtained from relatively short molecular dynamics simulations of the complex in water and of pure water based on the solution theory in the energy representation. We examined protein-protein complex model structures of two protein-protein complex systems, bovine trypsin/CMTI-1 squash inhibitor (PDB ID: 1PPE) and RNase SA/barstar (PDB ID: 1AY7), for which both complex and monomer structures were determined experimentally. For each system, we calculated the energies for the crystal complex structure and twelve generated model structures including the model most similar to the crystal structure and very different from it. In both systems, the sum of the conformational and solvation free energies tended to be lower for the structure similar to the crystal. We concluded that our energy calculation method is useful for selecting low energy complex models similar to the crystal structure from among a set of generated models.

Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2–89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2–89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8+ T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

Water Model Tuning for Improved Reproduction of Rotational Diffusion and NMR Spectral Density

A water model for molecular simulation was optimized to improve the reproduction of translational and rotational diffusion of pure water and proteins. The SPC/E(b) model was developed from the original SPC/E model with a slight increase of the O-H bond length of 1%. This tuning has significantly improved the translational and rotational diffusion when compared to the experimental values, whereas only small changes were observed in the other thermodynamic properties examined. The overall tumbling correlation times (τ(p)) from ubiquitin, protein G, bovine pancreatic trypsin inhibitor, and barstar C42/80A were successfully reproduced using the SPC/E(b) model. Calculated site-specific spectral densities of the main chain amide bond rotation in ubiquitin and protein G were in good agreement with those derived from nuclear magnetic resonance reduced spectral density mapping. The SPC/E(bT) model was also developed with temperature-dependent bond-length tuning to facilitate reproduction of the experimental τ(p) around room temperature.

Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.

High anisotropy and frustration: the keys to regulating protein function efficiently in crowded environments.

Highly anisotropic protein dynamics in equilibrium can be observed experimentally or through structural bioinformatics and molecular simulations. This anisotropic nature causes a response, to an external perturbation, along a small number of intrinsic large-amplitude directions as expected from the fluctuation-dissipation theorem. It is also key for controlling specific reactions as stochastic processes in macromolecular crowded environments. Protein anisotropy can be exploited for the calculation of physical properties, such as entropy, which can be employed for binding affinity studies. Energy frustration along soft modes including both global large-amplitude and localized small-amplitude movements is another key feature, as conformational transitions along soft modes, triggered by external perturbations such as the binding of other molecules, can act as a switch to control function.

Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature

The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight “zeppelin-shaped” dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in structured biomacromolecules

Life ticks as fast as how efficiently proteins perform their functional dynamics. Well-folded/structured biomacromolecules perform functions via large-scale intrinsic motions across multiple conformational states, which occur at timescales of nano-to milliseconds. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to gauge the time and sizes of these motions, though barely to their slowest ends. Here, we convert a computationally cheap elastic network model (ENM) into a molecular timer and sizer to gauge the slowest functional motions of proteins and ribosome. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener–Khintchine theorem (WKT) are used to define the “time-periods”, t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective “time-periods” are mapped to the eigenvalues (λENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 86.9λENM-1.9 and σ2(Å2) = 46.1λENM-2.5 are established allowing the characterization of the time scales of NMR-resolved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter. TOC Graphics

Protein–Ligand Dissociation Simulated by Parallel Cascade Selection Molecular Dynamics

We investigated the dissociation process of tri-N-acetyl-d-glucosamine from hen egg white lysozyme using parallel cascade selection molecular dynamics (PaCS-MD), which comprises cycles of multiple unbiased MD simulations using a selection of MD snapshots as the initial structures for the next cycle. Dissociation was significantly accelerated by PaCS-MD, in which the probability of rare event occurrence toward dissociation was enhanced by the selection and rerandomization of the initial velocities. Although this complex was stable during 1 μs of conventional MD, PaCS-MD easily induced dissociation within 100-101 ns. We found that velocity rerandomization enhances the dissociation of triNAG from the bound state, whereas diffusion plays a more important role in the unbound state. We calculated the dissociation free energy by analyzing all PaCS-MD trajectories using the Markov state model (MSM), compared the results to those obtained by combinations of PaCS-MD and umbrella sampling (US), steered MD (SMD) and US, and SMD and the Jarzynski equality, and experimentally determined binding free energy. PaCS-MD/MSM yielded results most comparable to the experimentally determined binding free energy, independent of simulation parameter variations, and also gave the lowest standard errors.

Binding free energy analysis of protein-protein docking model structures by evERdock

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.