Molecular Dynamics Analysis of Structural Factors Influencing Back Door Pi Release in Myosin

The back door has been proposed to be an exit pathway from the myosin active site for phosphate (Pi) generated by adenosine 5′-triphosphate hydrolysis. We used molecular dynamics simulations to investigate the interaction of Pi with the back door and the plausibility of Pi release via this route. Molecular dynamics simulations were performed on the Dictyostelium motor domain with bound Mg·adenosine 5′-diphosphate (ADP) and Pi, modeled upon the Mg·ADP·BeFx and Mg·ADP·Vi structures. Simulations revealed that the relaxation of ADP and free Pi from their initial positions reduced the diameter of the back door via motions of switch 1 and switch 2 located in the upper and lower 50-kDa subdomains, respectively. In neither simulation could Pi freely diffuse out the back door. Water molecules, however, could flux through the back door in the Mg·ADP·BeFx-based simulation but not in the Mg·ADP·Vi-based simulation. In neither structure was water observed fluxing through the main (front door) entrance. These observations suggest that the ability of Pi to leave via the back door is linked tightly to conformational changes between the upper and lower 50-kDa subdomains. The simulations offer structural explanations for 18O-exchange with Pi at the active site, and Pi release being the rate-limiting step in the myosin adenosine 5′-triphosphatase.