Investigating a back door mechanism of actin phosphate release by steered molecular dynamics, Proteins 35

ABSTRACT In actin-based cell motility, phos-phate (Pi) release after ATP hydrolysis is an essen-tial biochemical process, but the actual pathway of Pi separation from actin is not well understood. We report a series of molecular dynamics simulations that induce the dissociation of Pi from actin. After cleavage from ATP, the singly protonated phosphate (HPO422) rotates about the ADP-associated Ca21 ion, turning away from the negatively charged ADP towards the putative exit near His73. To reveal the microscopic processes underlying the release of Pi, adhesion forces were measured when pulling the substrate out of its binding pocket. The results suggest that the separation from the divalent cation is the rate-limiting step in Pi release. Protonation of HPO422 to H2PO42 lowers the electrostatic barrier during Pi liberation from the ion. The simulations revealed a propensity of charged His731 to form a salt bridge with HPO422, but not with H2PO42. His73 stabilizes HPO422 and, thereby, inhibits rapid Pi release from actin. Arg177 remains attached to Pi along the putative back door pathway, suggesting a shuttle function that facilitates the transport of Pi to a binding site on the protein surface. Protein