A point mutation in switch 1 alters the load dependence of phosphate rebinding to actomyosin.

Abstract

Myosin transduces chemical energy from ATP into mechanical work to generate force and/or motion. Key to this process is the coupling of the powerstroke and the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved remain unclear. We determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10 pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30 mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7 nm for the WT myosin. Repeating these experiments using a construct with a mutation in the Switch I region of the active site (S217A) increased myosin's sensitivity to load five-fold (d = 3.5 nm). Thus, these findings provide a quantitative measure of the force-dependence of Pi-rebinding to myosin and suggest that this effect involves the switch I element of the nucleotide-binding pocket.