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

Abstract

In actin-based cell motility, phosphate (P_i) release after ATP hydrolysis is an essential biochemical process, but the actual pathway of P_i separation from actin is not well understood. We report a series of molecular dynamics simulations that induce the dissociation of P_i from actin. After cleavage from ATP, the singly protonated phosphate (HPO₄²⁻) rotates about the ADP-associated Ca²⁺ ion, turning away from the negatively charged ADP towards the putative exit near His73. To reveal the microscopic processes underlying the release of P_i, 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 P_i release. Protonation of HPO₄²⁻ to H₂PO⁴⁻ lowers the electrostatic barrier during P_i liberation from the ion. The simulations revealed a propensity of charged His73⁺ to form a salt bridge with HPO₄²⁻, but not with H₂PO₄⁻. His73 stabilizes HPO₄²⁻ and, thereby, inhibits rapid P_i release from actin. Arg177 remains attached to P_i along the putative back door pathway, suggesting a shuttle function that facilitates the transport of P_i to a binding site on the protein surface. Proteins 1999;35:262–273. © 1999 Wiley-Liss, Inc.