A point mutation in the switch I region of myosin’s active site dramatically alters the load-dependence of phosphate-induced detachment from actin

Abstract

Myosin is a molecular motor responsible for generating the force and/or motion that drive many intracellular processes, from muscle contraction to vesicular transport. It is powered by its ability to convert the chemical energy, released from the hydrolysis of ATP, into mechanical work. The key event in the transduction process is the coupling of the force-generating powerstroke with the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved in this coupling remain unclear. Therefore, 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 0.10pN/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 30mM 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 0.7nm for the WT myosin. Repeating these experiments using a construct with a mutation (S217A) in a key region (Switch I) of the nucleotide-binding site increased myosin’s sensitivity to load five-fold (d = 3.5nm). Thus, these findings provide a quantitative measure of the force-dependent nature of Pi-rebinding to myosin’s active site and suggest that this effect involves the switch I element of the nucleotide-binding pocket. These findings, therefore, provide important new insights into the mechanisms through which this prototypical motor enzyme couples the release of chemical energy to the generation of force and/or motion.