Recent insights into the relative timing of myosin's powerstroke and release of phosphate

Abstract

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force‐generating powerstroke, and the release of phosphate (Pi) from the nucleotide‐binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High‐resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi‐release precedes the powerstroke. However, advances in functional assays, enabling sub‐millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

“…formation of a tight myosin-actin interface, actin-binding cleft closure, lever arm swing, and ATP hydrolysis products (phosphate and ADP) release. Despite extensive structural, biochemical, and single-molecule studies (see reviews 7,19,20 ), the causality and ordering of these events are difficult to characterize. Recent cryo-EM structures provide atomistic views of different myosin-actin isoforms at the strongly bound rigor state, [21][22][23][24] in which no nucleotide is bound to the active site.…”

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin

“…To continue the cycle, several events must occur, including Pi release, U50/L50 cleft closure, and lever arm rotation of the powerstroke. Although these events are tightly coupled, the timing of each remains debated (Llinas et al, 2015; Tang et al, 2021; Moretto et al, 2022; Debold, 2021). Nevertheless, during U50/L50 cleft closure, the upper and lower 50K domains rotate towards one another, resulting in stronger binding to F-actin(Milligan et al, 1990).…”

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

“…Indeed the motor domain of myosins, which includes the nucleotide-biding site and the actin-binding domain is highly conserved across the myosin family 9 . While these aspects are well known the mechanism underlying the motor domain's ability to couple the release of chemical energy from ATP with the generation of force and/or motion is still unclear [10][11][12][13][14] . This gap in knowledge limits the understanding of how this class of molecular motors accomplishes a multitude of intracellular tasks, which in turn limits our ability to identify the appropriate processes and structures to target for the development of effective treatments for a myriad of myosin-associated diseases 15,16 .…”

“…Hydrolysis of the ATP is thought to power the re-priming 19 of the long alpha helix, which serves as a lever arm, enabling production of the next force-generating powerstroke. The most crucial, but most poorly characterized, step in the transduction process is the coupled release of P i and the powerstroke 10,20,21 , this is therefore key to elucidating the mechanism of energy transduction by myosin 22 . In order to understand these steps investigators typically bathe myosin or muscle in excess amounts of P i to understand how it might rebind to the nucleotidebinding site and putatively reverse the P i -release step, and the powerstroke.…”

“…It is also unclear which regions or elements within myosin's active site might mediate the load-sensitivity of P i -rebinding to myosin's active site 10,12,20 . The release of P i is thought to occur through an opening created by a transient conformational change in the key, switch II, element of the nucleotide binding site, referred to as the back-door exit 30 .…”