FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

Abstract

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force generating lever arm in myosin V. We directly compared the FRET results with single molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), while our simulations suggest the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

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

“…To confirm that our analyses could detect a delay in the generation of the powerstroke if it occurred, we performed simulations of single binding events in which myosin bound to actin and either, rapidly generated a powerstroke (Figure 2b) or paused with a time constant of 30 ms before generating a step (Figure 2c). The time constant for P i -release was chosen to be consistent with the average measured rate of P i -release from the S217A construct from three different reports (Forgacs et al, 2009;Gunther et al, 2020;Llinas et al, 2015).…”

“…The loss of the hydroxyl group, thought to make contact with the gammaphosphate of ATP (Forgacs et al, 2009;Smith & Rayment, 1996), is hypothesized to impede the entry of P i into the exit tunnel (Llinas et al, 2015). Consistent with this hypothesis, actin-activated P irelease is 3-10-fold slower in this mutation compared to WT (Forgacs et al, 2009;Gunther et al, 2020;Llinas et al, 2015). In addition, x-ray crystal structures of myosin soaked in high concentrations of P i for 45 min show P i in the active site near ADP with myosin occupying a prepowerstroke state, supporting a P i -release gated powerstroke (Llinas et al, 2015).…”

“…The re-binding to actin triggers the release of P i and then ADP (Bagshaw & Trentham, 1974). The powerstroke is thought to occur close in time to the release of P i (Holmes & Geeves, 2000;Houdusse & Sweeney, 2016;Sweeney & Houdusse, 2010;Takagi, Shuman, & Goldman, 2004) from the active site, however the exact sequence of events is unclear and is currently the source of a vigorous debate within the field (Gunther et al, 2020;Llinas et al, 2015;Muretta, Rohde, Johnsrud, Cornea, & Thomas, 2015;Trivedi et al, 2015;Woody, Winkelmann, Capitanio, Ostap, & Goldman, 2019). The key unanswered question is which event occurs first the powerstroke or the release of P i from the active site (Llinas et al, 2015;Woody et al, 2019)?…”

Functional Role of Class III Myosins in Hair Cells

“…The hydrolysis of ATP occurs while myosin is detached from actin and allows the lever arm to be positioned in a pre-power stroke conformation (recovery stroke). When myosin with the hydrolyzed products in the active site binds to actin there is a transition into the strongly bound actomyosin state and a shift from a pre-power stroke to a post-power stroke state that is key to force generation (power stroke) as well as the acceleration of phosphate release ( Trivedi et al, 2015 ; Gunther et al, 2020 ). The release of ADP from actomyosin is required before another ATP cycle can occur.…”

Cardiomyopathy Mutations Impact the Power Stroke of Human Cardiac Myosin

“…It is well established that myosin, with the hydrolyzed products in its active site, associates weakly with actin, and then transitions into a strong actin-attached state to perform the power stroke. An initial actin-attached state has been proposed to be the pre-force generating intermediate before myosin transitions into the strong actomyosin state (11,52,53). The intermediate state is highly reversible (can rapidly detach from actin) but it is proposed to be stereospecific in nature (11).…”