Myosin's powerstroke occurs prior to the release of phosphate from the active site

Abstract

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 (P i ) 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 P i in its active site, by either elevating P i in solution or by introducing a mutation in myosin's active site (S217A) to slow P irelease from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s À1 ) and without a detectable delay, both in the absence and presence of 30 mM P i . The elevated levels of P i did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P i 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 P i release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P i still in its active site.

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

“…Single-molecule optical tweezers results 26,48 have favored a powerstroke-first model. The estimated time scale of powerstroke for cardiac myosin (1 ms 26 ) is independent of Pi concentration and faster than Pi release rate (ranging from a few ms to 60 ms 26,49 ).…”

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

“…To ensure that the assays and analyses they employed had the ability to detect a change in myosin's powerstroke, Scott et al (2021) simulated data assuming either a powerstroke-first or a P i -release first model and confirmed that if myosin bound to actin and paused before generating a powerstroke it would have been evident in their observations. Therefore, they concluded that their findings strongly favor a model in which myosin's powerstroke precedes the release of P i from its active site.…”

“…In a second independent test of this hypothesis, Scott et al (2021) determined the effect of increasing the P i concentration to 30 mM on the single molecule mechanics and kinetics using a wild-type myosin Va construct at 100 μM ATP. If P i -release gates the powerstroke, and therefore is released before the powerstroke, the elevated (P i ) should have reduced the amplitude of the powerstroke and/or slowed the rate the powerstroke.…”

“…Taking advantage of the mutation (S217A in myosin Va) used to support the new hypothesis (Llinas et al, 2015), two recent studies (Gunther et al, 2020; Scott, Marang et al, 2021) directly determined the effect of maintaining P i in the active on the size of and rate of development of a powerstroke at the single molecule level. Using a more traditional three‐bead laser trap assay, Scott et al (2021) directly examined the effect of the S217A mutation on the size and rate of the powerstroke in a single‐headed myosin Va construct.…”

“…Taking advantage of the mutation (S217A in myosin Va) used to support the new hypothesis (Llinas et al, 2015), two recent studies (Gunther et al, 2020; Scott, Marang et al, 2021) directly determined the effect of maintaining P i in the active on the size of and rate of development of a powerstroke at the single molecule level. Using a more traditional three‐bead laser trap assay, Scott et al (2021) directly examined the effect of the S217A mutation on the size and rate of the powerstroke in a single‐headed myosin Va construct. Despite dramatically slowing P i ‐release from the active site (Forgacs et al, 2009; Gunther et al, 2020; Llinas et al, 2015) the presence of this mutation did not reduce the size or rate of the powerstroke, suggesting that myosin rapidly generates a powerstroke with P i still in its active site.…”