Mechanochemical Coupling in the Myosin Motor Domain. II. Analysis of Critical Residues

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Abstract
An important challenge in the analysis of mechanochemical coupling in molecular motors is to identify residues that dictate the tight coupling between the chemical site and distant structural rearrangements. In this work, a systematic attempt is made to tackle this issue for the conventional myosin. By judiciously combining a range of computational techniques with different approximations and strength, which include targeted molecular dynamics, normal mode analysis, and statistical coupling analysis, we are able to identify a set of important residues and propose their relevant function during the recovery stroke of myosin. These analyses also allowed us to make connections with previous experimental and computational studies in a critical manner. The behavior of the widely used reporter residue, Trp501, in the simulations confirms the concern that its fluorescence does not simply reflect the relay loop conformation or active-site open/close but depends subtly on its microenvironment. The findings in the targeted molecular dynamics and a previous minimum energy path analysis of the recovery stroke have been compared and analyzed, which emphasized the difference and complementarity of the two approaches. In conjunction with our previous studies, the current set of investigations suggest that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely the general feature for mechanochemical coupling in many molecular motors. The fundamental strategies of examining both collective and local changes and combining physically motivated methods and informatics-driven techniques are expected to be valuable to the study of other molecular motors and allosteric systems in general. Molecular motors are inherently allosteric in nature because the small structural changes associated with the chemistry in the active site are propagated over a long distance and amplified into much larger conformational transitions. A fundamental challenge for understanding such processes concerns the identification of residues and interactions that dictate the propagation of the “mechanochemical coupling signals.” By combining a range of computational techniques based on different principles and assumptions, we have made a systematic attempt to identify these hotspot residues in the molecular motor myosin. Although each method has its limitations, the results from different analyses complement and reinforce each other, which makes it possible to meaningfully compare the results here to previous computational and experimental studies. Combined with results from the preceding paper [24], the current set of investigations suggests that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely a general feature for many molecular motors. The study also highlights the value of examining both collective and local changes as well as combining physically motivated methods and informatics-driven techniques for analyzing allosteric systems in general.