Mechanochemical coupling in myosin: a theoretical analysis with molecular dynamics and combined QM/MM reaction path calculations

To elucidate the detailed mechanism of ATP hydrolysis in myosin, molecular dynamics employing classical force field and reaction path analyses employing a combined quantum mechanical and molecular mechanical (QM/MM) potential were carried out. Although the QM/MM reaction path analyses have limited accuracy due to the lack of extensive conformational sampling, the present work showed that sensible energetics much closer to experimental measurements than previous computational studies can be obtained once the protein environment is included. In the two associative mechanisms studied here, the pathway that involves the conserved residue, Ser 236, as the proton relay group was found to have a lower rate-limiting barrier. However, it was also shown that if O2ç gets protonated, the mechanism without invoking any proton relay has only a slightly higher barrier and therefore may also contribute, especially in mutants such as Ser 236A. By performing calculations for two different motor domain conformations, it was shown that the mechanochemical coupling in myosin is mainly regulated by several residues in the switch I and switch II regions, such as Arg 238, Gly 457, and Glu 459. In particular, when the salt bridge between Arg 238 and Glu 459 is broken as in the prehydrolysis conformation of the motor domain, ATP hydrolysis is highly unfavorable energetically. In this conformation, Arg 238 is closer to ATP and therefore stabilizes the ATP state over the hydrolysis products. Moreover, without the salt bridge, the water structure in the active site is no longer stabilized to favor the in-line nucleophilic attack of the ç-phosphate. The results from the current work have general implications to other molecular motors that involve ATP hydrolysis, such as kinesin, F1-ATPase and Ca2+-ATPase, although more robust conclusions concerning the hydrolysis mechanism require more elaborate simulations that consider protein fluctuations and other possible protonation states of ATP and the hydrolysis products. I