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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.

Myosin is biological molecular motor that transduces the chemical energy ATP hydrolysis into force and/or motion to drives many forms of cellular motility from muscle contraction to cell division. While the transduction process has been extensively studied key details remain unclear. Most importantly the timing of the force-generating powerstroke relative to the release of phosphate (Pi), a product of ATP hydrolysis, is not clear and the source of intense debate. We examined the ability of single-headed myosin Va to generate a displacement (i.e. powerstroke) of an actin filament in a kinetic, fluorescent energy transfer (FRET) and single molecule laser trap assays while maintaining Pi in its catalytic site by introducing a mutation that impedes Pi-release from the catalytic site (S217A). Kinetic experiments confirmed that the Pi-release rate was slowed ~5-fold by the S217A substitution, and FRET experiments suggested that the mutation slowed myosin’s rate of attachment to actin. At the single molecule level Wild type myosin Va (WT) generated a 7 nm powerstroke at a rate of ~600/s and the S217A myosin generated a statistically similar size powerstroke (8nm) at a similar rate (600/s) despite impeding the release of Pi from the active site. These findings are consistent with myosin Va generating a powerstroke while Pi is still in the catalytic site, and therefore challenge the hypothesis that Pi-release gates, and therefore precedes, the powerstroke. Our findings imply that myosin’s key mechanical event is triggered by binding to the actin filament, and not by the biochemical event of Pi-release. This provides important new insight into the mechanism of energy transduction by myosin, and evolutionarily related motor proteins.

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (Pi ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining Pi in its active site, by either elevating Pi in solution or by introducing a mutation in myosin's active site (S217A) to slow Pi -release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s-1 ) and without a detectable delay, both in the absence and presence of 30 mM Pi . The elevated levels of Pi did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that Pi rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower Pi release rate. These findings provide direct evidence that myosin Va generates a powerstroke with Pi still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of Pi from the active site.