尾嶋 拓

Precise regulation of protein kinase activity is crucial in cell functions, and its loss is implicated in various diseases. The kinase activity is regulated by interconverting active and inactive states in the conformational landscape. However, how protein kinases switch conformations in response to different signals such as the binding at distinct sites remains incompletely understood. Here, we predict the binding mode for the peptide substrate to Src tyrosine kinase using enhanced conformational sampling simulations (totaling 24 μs) and then investigate changes in the conformational landscape upon substrate binding by conducting unbiased molecular dynamics simulations (totaling 50 μs) initiated from the apo and substrate-bound forms. Unexpectedly, the peptide substrate binding significantly facilitates the transitions from active to inactive conformations in which the αC helix is directed outward, the regulatory spine is broken, and the ATP-binding domain is perturbed. We also explore an underlying residue-contact network responsible for the allosteric conformational changes. Our results are in accord with the recent experiments reporting the negative cooperativity between the peptide substrate and ATP binding to tyrosine kinases and will contribute to advancing our understanding of the regulation mechanisms for kinase activity.

<jats:title>Abstract</jats:title><jats:p>Multistate Bennett acceptance ratio (MBAR) works as a method to analyze molecular dynamics (MD) simulation data after the simulations have been finished. It is widely used to estimate free-energy changes between different states and averaged properties at the states of interest. MBAR allows us to treat a wide range of states from those at different temperature/pressure to those with different model parameters. Due to the broad applicability, the MBAR equations are rather difficult to apply for free-energy calculations using different types of MD simulations including enhanced conformational sampling methods and free-energy perturbation. In this review, we first summarize the basic theory of the MBAR equations and categorize the representative usages into the following four: (i) perturbation, (ii) scaling, (iii) accumulation, and (iv) full potential energy. For each, we explain how to prepare input data using MD simulation trajectories for solving the MBAR equations. MBAR is also useful to estimate reliable free-energy differences using MD trajectories based on a semi-empirical quantum mechanics/molecular mechanics (QM/MM) model and ab initio QM/MM energy calculations on the MD snapshots. We also explain how to use the MBAR software in the GENESIS package, which we call <jats:italic>mbar_analysis</jats:italic>, for the four representative cases. The proposed estimations of free-energy changes and thermodynamic averages are effective and useful for various biomolecular systems.</jats:p>

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins in crowded solutions are aggregated with each other too strongly. To optimize the balance, Lennard-Jones (LJ) interactions between protein and water are often increased about 10% (with a scaling parameter, lambda = 1.1) from the existing force fields. Here, we explore the optimal scaling parameter of protein-water LJ interactions for CHARMM36m in conjunction with the modified TIP3P water model, by performing enhanced sampling MD simulations of several peptides in dilute solutions and conventional MD simulations of globular proteins in dilute and crowded solutions. In our simulations, 10% increase of protein-water LJ interaction for the CHARMM36m cannot maintain stability of a small helical peptide, (AAQAA)(3) in a dilute solution and only a small modification of protein-water LJ interaction up to the 3% increase (lambda = 1.03) is allowed. The modified protein-water interactions are applicable to other peptides and globular proteins in dilute solutions without changing thermodynamic properties from the original CHARMM36m. However, it has a great impact on the diffusive properties of proteins in crowded solutions, avoiding the formation of too sticky protein-protein interactions.