Step-wise Hydration of Magnesium by Four Water Molecules Precedes Phosphate Release in a Myosin Motor

Abstract

Molecular motors, such as myosin, kinesin, and dynein, convert the energy released by the hydrolysis of ATP into mechanical work, which allows them to undergo directional motion on cytoskeletal tracks. This process is achieved through synchronization between the catalytic activity of the motor and the associated changes in its conformation. A pivotal step in the chemomechanical transduction in myosin motors occurs after they bind to the actin filament, which triggers the release of phosphate (Pi, product of ATP hydrolysis) and the rotation of the lever arm. Here, we investigate the mechanism of phosphate release in myosin VI, which has been debated for over two decades, using extensive molecular dynamics simulations involving multiple trajectories each several s long. Because the escape of phosphate is expected to occur on time-scales on the order of milliseconds in myosin VI, we observed Pi release only if the trajectories were initiated with a rotated phosphate inside the nucleotide binding pocket. The rotation provided the needed perturbation that enabled successful expulsions of Pi in several trajectories. Analyses of these trajectories lead to a robust mechanism of Pi release in the class of motors belonging to the myosin super family. We discovered that although Pi populates the traditional \"back door\" route, phosphate exits through various other gateways, thus establishing the heterogeneity in the escape routes. Remarkably, we observe that the release of phosphate is preceded by a step-wise hydration of the ADP-bound magnesium ion. In particular, the release of the anion occurred only after four water molecules hydrate the cation (Mg2+). By performing comparative structural analyses, we suggest that the hydration of magnesium is the key step in the phosphate release in a number of ATPases and GTPases that share a similar structure in the nucleotide binding pocket. Thus, nature may have evolved hydration of Mg2+ by discrete water molecules as a general molecular switch for Pi release, which is a universal step in the catalytic cycle of many machines which share little sequence or structural similarity.