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. Author manuscript; available in PMC: 2022 Aug 3.
Published in final edited form as: Cytoskeleton (Hoboken). 2022 Mar 21;78(9):448–458. doi: 10.1002/cm.21695

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

Edward P Debold 1
PMCID: PMC9347231  NIHMSID: NIHMS1824755  PMID: 35278035

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.

Keywords: force-generation, myosin, phosphate-release, powerstroke

1 |. INTRODUCTION

Myosin and related molecular motors generate force and/or motion by converting chemical energy from ATP into mechanical work (Vale, 2000). This force- and motion-generating capacity enables myosin to drive a myriad of critical cell processes, from muscle contraction to vesicular transport to cytokinesis (Vale, 2003). Its force- and motion-generating capacity is due to its ability to generate a powerstroke, a large rotation of a long alpha helical coil in the motor domain, commonly referred to as the lever arm (Rayment et al., 1993). This powerstroke occurs close in time with the release of Pi from the nucleotide-binding site, an event thought to result in a large release of free energy (Eisenberg & Hill, 1985; Howard, 2001; Inoue et al., 1989). The large release of energy suggested that the mechanical powerstroke is closely associated with, if not tightly coupled to, the biochemical event of Pi-release. Indeed, early models of myosin cross-bridge cycle depicted these two events occurring simultaneously upon binding to actin (Bagshaw & Trentham, 1974; Chock et al., 1979; Lymn & Taylor, 1971). However recent technological advances, enabling researchers the ability to observe these events with sub-millisecond time resolution, are revealing that the two events are temporally distinct (Capitanio et al., 2012; Muretta, Rohde, Johnsrud, Cornea, & Thomas, 2015). These recent findings have sparked a vigorous debate over which event occurs first, Pi-release or the powerstroke. Answering this question will help understand which event provides the energy for the powerstroke, and thus, the generation of force and/or motion by myosin. This review provides some historical context on the question before highlighting some key recent findings that shed new light on this fundamental question of how this prototypical molecular motor transduces energy.

1.1 |. The powerstroke and Pi-release occur rapidly upon binding to actin

During its cross-bridge cycle myosin goes through mechanical transitions (e.g., the powerstroke and actin binding and unbinding) and biochemical transitions (e.g., ATP hydrolysis and Pi- and ADP-release), as it cyclically interacts with actin and generates force and/or motion. In a simplified model of the cross-bridge cycle (Figure 1), ATP binding to the rigor state induces conformational changes that cause myosin to dissociate from actin (Geeves & Holmes, 2005). ATP is then hydrolyzed to ADP and Pi which remain in the active site while myosin is detached from actin. Either right before (Geeves & Holmes, 2005) or during the hydrolysis step, myosin’s lever arm is re-primed (Steffen & Sleep, 2004) to the pre-powerstroke state, getting it ready for the next powerstroke. Re-binding to actin, once a strong, stereo-specific attachment is formed, rapidly triggers the release of Pi from the active site, which is followed by ADP-release (Bagshaw & Trentham, 1974; Lymn & Taylor, 1971). These biochemical events are coupled to the mechanical events of binding to and unbinding from actin, as well as the generation of a powerstroke (Geeves & Holmes, 2005; Sweeney & Houdusse, 2010). The powerstroke results from small conformational changes within the ATP-binding site being amplified in the converter region to cause the large, 60–70°, rotation of the lever arm (Geeves & Holmes, 2005; Preller & Holmes, 2013; Rayment, Holden, et al., 1993; Sweeney & Houdusse, 2010). The powerstroke is thought to occur close in time with the release of Pi (Takagi, Shuman, & Goldman, 2004) from the active site and both events occur rapidly after binding to actin (Bagshaw & Trentham, 1974), but which event occurs first? The answer to this question lies at the heart of the long-standing quest to understand the molecular basis of force-generation by muscle myosin (Huxley & Simmons, 1971; Linari et al., 2015; Piazzesi et al., 2002; Veigel, Molloy, Schmitz, & Kendrick-Jones, 2003). It has even broader implications because this transduction pathway is highly conserved among molecular motors; therefore, the knowledge is critical for a detailed understanding of how related motor proteins transduce chemical energy into mechanical work (Vale, 1996, 2003).

FIGURE 1.

FIGURE 1

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A simplified model of myosin’s cross-bridge cycle. Schematic depicts key steps in myosin’s ATPase/cross-bridge cycle. The shaded boxes indicate the two proposed models for the relative timing of the powerstroke and Pi-release from the active site

This has been a challenging question to answer for two key reasons: firstly, because the actomyosin complex is not amenable to crystallization, which limits the structural detail needed to see actin-induced changes in the active site that lead to force-generation and Pi-release (Preller & Holmes, 2013); and secondly because the speed of the powerstroke (<1 ms) has, until recently (Capitanio et al., 2012), exceeded the time resolution of traditional single molecule laser trap assays. However, recent advances in both structural biology and single molecule biophysical techniques have allowed for new insights into this compelling question (Gunther et al., 2020; Llinas et al., 2015; Muretta et al., 2015; Trivedi et al., 2015; Woody, Winkelmann, Capitanio, Ostap, & Goldman, 2019).

2 |. THE STATE OF KNOWLEDGE PRIOR TO 2012

2.1 |. Early insights from functional assays

Chemically skinned single skeletal muscle fiber preparations were initially used to determine the relative timing of force-generation and Pi-release. These efforts demonstrated that force developed much faster than the measured rate of Pi-release (Z. H. He et al., 1997; J. Sleep, Irving, & Burton, 2005). However, the relatively large diameter of a muscle fiber limited the speed of the diffusion of reagents and reporters of Pi-release; therefore, it was not clear if force-generation preceded Pi-release from the active site, or if the appearance of Pi in solution and/or the rate of binding to the reporter occurred more slowly than the generation of force. Caged-phosphate compounds were used to attempt to overcome this limitation by allowing for the rapid release of mM amounts of Pi in an isometrically contracting single muscle fiber (Dantzig, Goldman, Millar, Lacktis, & Homsher, 1992). Following the release of the caged Pi a short but measurable delay was detected before isometric force was reduced. In addition, the rate and amount of the reduction in force displayed a strong dependence on the [Pi]. These two observations led the authors to propose a model in which myosin first strongly binds to actin generates force and a powerstroke, before subsequently releasing Pi. Following the release of Pi, in this model, actomyosin transiently exists in two ADP-bound states, separated by an isomerization (Hibberd, Dantzig, Trentham, & Goldman, 1985; J. A. Sleep & Hutton, 1980). The first of these states is thought to be prolonged by a resistive load (Takagi et al., 2004; Veigel et al., 2003). Pi can rebind to this pre-isomerization, strained state and induce the reversal of the force-generating steps, including the powerstroke (Sellers & Veigel, 2010). Thus, these and similar findings were thought to be consistent with the powerstroke preceding the release of Pi.

However, this interpretation of these data is not universally accepted (Månsson, Rassier, & Tsiavaliaris, 2015; Månsson, 2019; Offer & Ranatunga, 2020; D. A. Smith, 2014), and indeed, has been challenged recently by efforts to model these and related findings from muscle fibers, using kinetic models that treat Pi-release and the powerstroke as distinct and separate events (Månsson, 2019; Offer & Ranatunga, 2020; D. A. Smith, 2014). The most direct test of the two hypotheses evaluated two models in which Pi-release was made to occur either before or after the powerstroke (Offer & Ranatunga, 2020). Both models seemed to reproduce the effects on the shortening region of the force velocity-relationship, but a Pi-release-first model seemed to reproduce the effects of Pi on isometric force and tension transients in response to lengthening better than a powerstroke-first model. While the differences between the two models were not statistically quantified, nor a detailed sensitivity analysis performed, the authors concluded that the Pi-release-first model reproduced the effect of Pi on the examined contractile properties better than a powerstroke-first model. An important caveat with this and similar models is that some of the fit parameters may be inconsistent with experimental observations. For example, in this report (Offer & Ranatunga, 2020) the rate of Pi-release changed dramatically during refinement of the model, increasing from a rate of 70/s, a value taken from measurements in solution (White et al., 1997), to 500/s for the Pi-release-first model and over 6,000/s for the powerstroke-first model. These values are two to three orders of magnitude higher than the measured values for myosin II in solution (White et al., 1997). This disparity, as the authors suggest, confirms the need to perform a complete sensitivity analysis on these types of models before definitive conclusions can be drawn on the order of events.

Ultimately, the spatial and temporal resolution of the single muscle fiber assays cannot provide direct evidence of how, and when, a single myosin molecule goes through its mechanical and biochemical transitions; thus, these findings cannot provide a direct answer to the question of the timing of these events at the level of an individual myosin molecule. This has led researchers to employ more direct methods to address this question (Muretta et al., 2015; Trivedi et al., 2015), including single molecule assays (Finer, Simmons, & Spudich, 1994).

2.2 |. Structural evidence suggests Pi-release precedes the powerstroke

The original X-ray structure of fast skeletal myosin, obtained in the absence of ATP, demonstrated that the lever arm was not only in a post-powerstroke position but also revealed a large, open cleft in the upper 50 kDa domain (Rayment et al., 1993). Attempts to fit this structure into the envelop of an actin filament decorated with rigor myosin from low-resolution cryo-EM images revealed that the best fit was obtained only when this cleft was tightly closed (Rayment et al., 1993). In this confirmation the switch II loop, within the nucleotide-binding site, was found to be in an open state, a configuration with a low affinity for the gamma-Pi of ATP, presumably facilitating its release from the active site. These structural observations led to the hypothesis that the opening and closing of the actin-binding cleft reciprocally modulates myosin’s affinity for Pi. Specifically, when the actin-binding cleft is open, an invariant glycine residue in the switch II element is in a position that contacts the gamma-Pi, enabling hydrolysis. However, the formation of a strong-bond with actin induces closure of the actin-binding cleft, which reciprocally opens switch II, providing a mechanism for Pi release from the active site via a “back-door” exit (Holmes & Geeves, 2000; Yount et al., 1995). These events were postulated to be coupled and therefore the trigger that initiates the start of the powerstroke. Thus, this was the first report to suggest that the release of Pi from the active site occurs before the powerstroke, implying that Pi-release gates the powerstroke (Sweeney & Houdusse, 2010).

However, this crystal structure of myosin (Figure 2) was obtained in the absence of an actin filament and in the absence of a nucleotide; thus, these ideas needed to be more directly tested. Therefore, in subsequent studies myosin crystal structures were generated in the presence of various nucleotide analogs to “trap” myosin in putative intermediate states of the cross-bridge cycle. Crystals of single-headed S1 myosin from Dictyostelium discoidem grown with beryllium or aluminum fluoride and MgADP in the active site, serving as analogs of nucleotide bound states, demonstrated that switch II was close enough to the Pi-analog to support hydrolysis. This was referred to as the “closed” position (Fisher et al., 1995) of switch II. Switch II also appears to be in a “closed” position in crystals grown with MgADP and vanadate, which is considered a more appropriate analog of Pi (Smith & Rayment, 1996). With switch II in a closed position the converter and the truncated version of the light chain binding-domain are in a pre-powerstroke position, and in an ADP-Pi like biochemical state. Thus, while the Pi is held in the active site the lever arm remains in a pre-powerstroke position. The motions in the active site are thought to be coupled to rotation of the lever arm via an alpha-helix connecting switch II with the converter domain, termed the relay helix. Thus, this provided a potential structural basis for how the actin-binding affinity was coupled to the occupancy of Pi in the active site and the position of the lever arm. However, it is still unclear which motions trigger which event, that is does cleft closure trigger Pi-release which then permits lever arm rotation, or does closure of the actin-binding cleft trigger the rotation of the lever arm which then allows Pi to be released from the active site? This question of the relative timing of these rapid and dynamic events has been difficult to answer using images of static protein structures in the absence of actin. Indeed, there is ample evidence that the structural elements within myosin are likely fluctuating between the extreme positions observed in rigor versus the transition states (Conibear et al., 2003; Málnási-Csizmadia et al., 2001; Ostap et al., 1995; Yengo et al., 2002). These findings suggest that techniques that can track myosin’s micro-to milli-second time scale structural changes are needed to compliment the information from high-resolution static structures to understand the timing and order of key events in the cycle. In this review, we highlight some of the recent findings that have measured myosin’s structural dynamics in solution taking advantage of the high time and spatial resolution biophysical techniques to answer this fundamental question.

FIGURE 2.

FIGURE 2

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(a) A high-resolution crystal structure of myosin (myosin V) showing key elements and regions of myosin (image modified from Houdusse & Sweeney, 2016), reprinted with permission. (b) An enlargement of the active site in the presence of a ADP and vanadate (an analog of Pi). Shown as the PiR1 state with the putative Pi-release tunnel highlighted. Image from Llinas et al. (2015), reprinted with permision. Note residue R203 at the mouth of the tunnel, which is 217 in myosin Va. (c) Superimposed crystal structures of myosin in the pre- and post-powerstroke states (ADP and rigor), image from Wulf et al. (2016), reprinted with permission

Determining the relative timing of these key events may also have important clinical implications for the development of therapies to treat muscle disorders and weakness. For example, some of the most promising lead compounds designed to treat different forms of heart failure appear to target force-generating and Pi-release processes in cardiac myosin (Malik et al., 2011; Scellini et al., 2021). However, it is becoming clear that a complete understanding of the mechanisms responsible for the generation of force and Pi-release are required to understand the mechanism of action of these compounds. Indeed, the mechanism of action of one promising new drug for heart failure, currently in phase III clinical trials (Felker et al., 2021), is still unclear because it depends on knowing whether Pi-release or the powerstroke occurs first in the cycle. Kinetic studies suggest that the compound (omecamtiv mecarbil, OM) accelerates Pi release (Malik et al., 2011; Rohde et al., 2017), but its effect on the powerstroke remains equivocal (Planelles-Herrero et al., 2017; Woody et al., 2018).

Evidence from structural studies suggests that OM traps myosin in a pre-powerstroke state which accelerates the steps leading to Pi-release, which allows the powerstroke to occur, when OM unbinds from myosin, letting it proceed through ADP-release and ATP-binding and detachment from actin (Planelles-Herrero et al., 2017). Thus, under this mechanism the drug putatively enhances force by accelerating myosin’s rate of attachment to actin. In contrast, single molecule observations suggest that OM prevents the powerstroke from occurring (Woody et al., 2018). The authors of this study contend that the drug enhances force by increasing activation of the thin filament via strong-binding activation of the thin filament (Kad et al., 2005), and they demonstrate how this mechanism can explain prior observations (Woody et al., 2018). If this mechanism is correct the enhancement of force in cardiac muscle would be most effective when the drug affects a small fraction of the myosin molecules, higher concentrations may actually reduce force and power. If instead OM accelerates the rate of attachment (Planelles-Herrero et al., 2017) much higher doses might be needed to enhance function in a failing heart. Therefore, knowledge of the relative timing of Pi-release and the powerstroke could have important practical implications for clinicians as well as providing fundamental knowledge to basic scientists.

3 |. RECENT FINDINGS

3.1 |. FRET and single molecule laser trap assays suggest that the powerstroke precedes Pi-release

Advances in Föster Resonance Energy Transfer (FRET) assays have offered researchers a powerful new approach to determine the timing of the powerstroke relative to Pi-release, by providing direct real-time detection of the structural dynamics during myosin’s mechanochemical cycle (Muretta et al., 2015; Trivedi et al., 2015). In the typical experimental arrangement one fluorescent probe is placed on myosin’s regulatory light chain, serving as the donor, while a fluorescently labeled nucleotide serves as the acceptor in the FRET assay (Figure 3). This approach enables direct monitoring of the position of the lever arm and the occupancy of the nucleotide-binding site, with nanometer spatial and millisecond time resolution. Because myosin’s strong-binding to actin accelerates both the release of Pi and the powerstroke, the rate of Pi-release can be determined using the rapid reporting phosphate-binding protein in a parallel set of experiments and then the two rates are compared. These assays are performed using a stopped-flow apparatus, where the myosin is pre-incubated for ATP to ensure it is in an M.ADP.Pi state before it is rapidly mixed with actin, during which the rate of the powerstroke and Pi release are measured (Muretta et al., 2015; Trivedi et al., 2015). Two rates of the powerstroke rates are typically observed; one rapid rate occurring at a maximum of 350–500/s and a second slower one at a rate of 20–35/s (Trivedi et al., 2015). The more rapid rate is consistent with the rapid and large rotation of the lever arm that occurs upon strong-binding to actin, while the second slower rate is thought to correspond to the secondary powerstroke or “hitch” that is associated with the release of ADP (Dominguez et al., 1998). Both phases of the powerstroke are dependent on the actin concentration, with the amplitude of the fast phase dominating the amplitude of the signal.

FIGURE 3.

FIGURE 3

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(a) Structure of actin (yellow) and myosin (red/green) used in FRET experiments to determine the rate of the powerstroke. The distance between the acceptor and donor fluorophores is depicted for the pre- (M**) and post-stroke (M*) states. The distribution of the distance, in nanometers (nm), between the donor/acceptor pair is overlayed with fit lines of red and green. (b) The rate (kobs) of the fast phase of Pi-release versus the actin concentration used in the transient kinetic experiments. (c) The rate of the fast phase of the powerstroke versus the actin concentration. Two sets of values (solid and hollow red circles and solid and dotted line fits) were obtained for the rate of the fast phase of powerstroke based on the two distinct methods used to estimate this rate. The maximum rate of Pi-release was ~25/s while the maximum rate of the powerstroke was ~350/s. Figures modified with Muretta et al. (2015) reprinted with permission

Using fast skeletal muscle myosin II (HMM), Muretta et al. (2015) observed that the rapid rotation of myosin’s lever arm (fast phase of the powerstroke) occurred at a maximum measure rate of >350/s while, in the parallel experiment, actin-activated Pi-release saturated at only ~25/s (Figure 3). Thus, these findings suggested that the powerstroke occurs at a rate 10 times faster than Pi-release. They were careful to acknowledge that the faster rate of the powerstroke does not necessitate that it precedes Pi-release, so they modeled the results to help delineate between a powerstroke- or Pi-release-first model. These model simulations revealed that the data were very poorly fit by a Pi-release-first model, but were well-fit by a powerstroke-first model (Muretta et al., 2015). This suggests that once strongly bound to actin, myosin rapidly generates a powerstroke before releasing Pi at a much slower rate.

Similar results were obtained using single-headed myosin Va using a FRET pair on the n-terminus and another on the light chain bound to the first IQ motif (Trivedi et al., 2015). This experiment represented an important new test of the order of events because myosin Va is known to have a much faster actin-activated rate of Pi-release than myosin II (Baker et al., 2004; De La Cruz et al., 1999; Rosenfeld & Sweeney, 2004). Despite having a much faster rate of Pi-release of than myosin II at 200/s, the FRET-based assays revealed that lever arm rotation (i.e., the powerstroke) still occurred nearly twice as fast as Pi-release, reaching a maximum rate of 500/s. Computer simulations were again used to help delineate between the two models, and the results suggested that the data were most consistent with a model in which the powerstroke precedes Pi-release. Thus, these FRET-based studies demonstrate that two different classes of myosins appear to go through the same order of events, with strong-actin binding inducing a rapid force-generating powerstroke before Pi is released from myosin.

The other functional assay which has recently provided novel insight into the timing of these events has been the three-bead single molecule laser trap assay. The initial findings from this type of assay demonstrated that myosin rapidly generates a powerstroke upon binding to the actin filament (Finer et al., 1994), suggesting that myosin may generate a powerstroke before Pi is released. However, the time resolution of the earliest version of this assay was limited to ~2–10 ms, much slower than the putative rate of the powerstroke of <1 ms (Dunn & Spudich, 2007). To improve the time resolution, Capitanio et al. (2012) altered the assay by actively oscillating the traps and the actin filament in a sawtooth pattern so that myosin experienced a load almost instantaneously upon binding to the actin filament (Figure 4). Combined with a newly a developed analysis, this technique increased the time resolution by an order of magnitude, from a few milliseconds down to tens of microseconds. With this improve time resolution the displacement caused by the powerstroke can be directly visualized, a transformational development for the field. In addition, this advance afforded the ability to directly observe the weak, non-productive interactions that occur without generating a powerstroke. Thus, two populations of binding events are observed in this experiment; short events (<1.5 ms) with no powerstroke whose lifetime is ATP-independent (weak-binding interactions), and long events (>1.5 ms) that generate a powerstroke and have an ATP-dependent lifetime (i.e., productive-binding events).

FIGURE 4.

FIGURE 4

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(a) A schematic of the ultra-fast single molecule laser trap assay used to determine the rate of the myosin’s powerstroke (i.e., working stroke). The optical traps are oscillated with acoustoptic deflectors (AOD) to apply alternating positive and negative forces onto the bead-and-bead assembly. Bead position and therefore the position of the actin filament is tracked using quadrant photodiodes (QPD). (b) Upon binding to the actin filament the specified load (e.g., −2pN) is applied to the myosin molecule causing a pause in the sawtooth wave. (c) An expanded image of an individual actomyosin-binding event showing the short pause before the powerstroke is generated (stroke). Events are ended with unbinding typically caused by ATP-binding. (d) An ensemble average for binding events temporally aligned to show the average size (4–5 nm) and rate of the powerstroke. Image modified from (Capitanio et al., 2012), reprinted with permission. (e) A plot of the rate of the powerstroke (stroke) as a function of the applied load in the ultra-fast laser trap assay. Image from Woody et al. (2019), reprinted with permission

Using single-headed fast skeletal myosin II, Capitanio et al. (2012) observed that during the long events myosin briefly paused in a pre-powerstroke state before generating a powerstroke of 5 nm in less than 1.5 ms (Figure 4). This observed rate of the powerstroke is roughly an order of magnitude faster than the observed rate of Pi-release from myosin II (He et al., 1998; J. Sleep et al., 2005), suggesting that the powerstroke occurs prior to Pi-release. This represented a significant advance in the time resolution of functional assays of myosin; however, Pi-release was not directly monitored in these experiments, nor was the concentration of Pi in the buffer manipulated, so the timing of its release relative to the powerstroke was not directly determined from these findings.

In a subsequent study, specifically designed to determine the relative timing of the powerstroke and Pi-release, Woody et al. (Woody et al., 2019) performed a similar series of experiments using cardiac muscle myosin II (HMM). Consistent with prior findings using fast skeletal myosin (Capitanio et al., 2012), the longer duration binding events myosin generated a powerstroke at a rate of 700–5,250/s after binding to actin. Importantly, neither the rate nor the size of the powerstroke of the long events was affected by the addition of 10 mM Pi to the experimental buffer (Woody et al., 2019), suggesting that the powerstroke precedes Pi-release in cardiac myosin. Indeed, given the fast rate of the powerstroke, for these observations to be consistent with a model in which Pi release occurs prior to the powerstroke, Pi would need to be released at a rate of >5,000/s, two orders of magnitude faster than the observed rate of Pi-release in cardiac myosin (Tang et al., 2021). Furthermore, if Pi-release “gated” the powerstroke then the expectation would be that the elevated Pi in the buffer would have slowed the rate of the powerstroke, which was not observed in the data records. Since there was no observed effect on the rate of the powerstroke, and a Pi-release rate of >5,000/s seemed implausible, the authors concluded that the findings strongly suggest that the powerstroke precedes Pi-release.

However, a caveat with these findings was that the amount of short and intermediate duration binding events (events that did not generate a powerstroke) increased in the presence of elevated Pi, at the lowest applied forces (1.5 and 2.25 pN). This was interpreted by the authors as being consistent with an increase in the frequency of events detaching from the weakly bound state. However, this has also been interpreted as resulting from Pi rebinding to actomyosin while it is in an ADP-bound state, prior to the generation of the powerstroke and preventing the powerstroke from occurring, before accelerating detachment from actin (Robert-Paganin et al., 2020). The latter interpretation would be consistent with a model where Pi release occurs prior to the powerstroke (Llinas et al., 2015).

Further complicating the interpretation of these data is ambiguity over the actomyosin state to which the Pi likely rebound, resulting from the low ATP (1 μM) used in the experimental buffer (Woody et al., 2019). At this ATP and Pi concentration (10 mM), Pi likely rebound to a significant proportion of actomyosin heads in the rigor state, forming an off-pathway AM.Pi state that prolongs the rigor lifetime (Amrute-Nayak et al., 2008; Debold et al., 2011) instead of rebinding to an AM. ADP state to form an AM.ADP.Pi state. This occurs because Pi competes with ATP for binding to the empty nucleotide-binding-pocket (Amrute-Nayak et al., 2008). Indeed, the prolongation of the rigor state by Pi may explain the 2-fold reduction in the detachment rates for the slowest rate (representing events that complete a powerstroke) in the presence of Pi (Woody et al., 2019). Thus, these biophysical assays seem to suggest that the powerstroke may precede the release of Pi from the active site, challenging the view based on structural evidence, but ambiguity around the classification of the duration of binding events, and the actomyosin state to which Pi rebound temper the conclusions.

3.2 |. New crystal structures suggest release of Pi from the active site is faster than its release into solution

To attempt to resolve the conflict between the functional and structural evidence, Llinas et al. (2015) crystallized myosin in the presence of Pi to reveal the structures during Pi-release and rebinding. Myosin VI crystals were grown in the presence of MgADP and then soaked in a cryo-protectant with high levels of Pi (25–100 mM) for increasing periods of time before rapidly freezing the samples and using them to generate new X-ray structures. Freezing the samples as quickly as possible produced a structure with Pi near the exit of the Pi-release tunnel (Figure 2), which they termed the PiR1 state. A delay of just a few seconds resulted a structure with Pi close to ADP in the nucleotide-binding region (PiR2, i.e., still in the active site). While this construct lacked a lever arm domain, the configuration of the converter domain in both structures was unchanged, that is, consistent with a post powerstroke state. However, longer delays before freezing revealed that Pi diffused all the way back into the active site close to ADP, and when it did the converter region returned to a position consistent with a pre-powerstroke configuration. These observations led the authors to hypothesize that Pi leaves the active site very rapidly, and prior to the powerstroke, but stalls in the Pi-release tunnel before it is released into solution. Thus, they proposed that Pi-release from the active site occurs much more rapidly than it appears in solution. If correct this would provide an explanation for why Pi-release into solution is observed as being slower than the rate of the powerstroke in functional assays (Muretta et al., 2015; Trivedi et al., 2015). Therefore, Pi-release from the active site may occur before the powerstroke but its appearance in solution may happen after the powerstroke.

As an indirect test of this hypothesis, Llinas et al. (2015) introduced a mutation into switch I of the active site specifically designed to slow Pi-release from the active site by impeding it from entering the exit tunnel (S217A in myosin Va). The loss of the hydroxyl group, thought to make contact with the gamma-phosphate of ATP, is hypothesized to impede the entry of Pi into the Pi-release tunnel (Llinas et al., 2015). Consistent with this hypothesis actin-activated Pi-release was 3- to 10-fold slower in this mutation compared to WT (Forgacs et al., 2009; Gunther et al., 2020; Llinas et al., 2015). S217A also appears to slow the rate of the weak to strong-binding transition, which at high ionic strengths and sub-saturating actin concentrations likely contributes to the slowed rate of Pi-release (Gunther et al., 2020), but even at saturating actin concentrations the Pi-release rate remains slow in this mutant, providing strong evidence that it also slows Pi-release even once strongly bound to actin (Forgacs et al., 2009).

Importantly, the effect of S217A on myosin’s powerstroke was not directly tested in this investigation (Forgacs et al., 2009), so it was not clear if it affected the rate or size of the powerstroke, and this new hypothesis makes very testable predications about how a single myosin molecule should behave if Pi is maintained in the active site. For example, a myosin construct with an mutation that slows the entry of Pi into the Pi exit tunnel (e.g., S217A; Forgacs et al., 2009; Llinas et al., 2015) should dramatically delay, or even prevent, myosin from generating a powerstroke once it strongly binds to actin. Similarly, maintaining Pi in the active site, by exposing it to high concentrations of Pi, should delay or even prevent the powerstroke, if Pi-release “gates” the powerstroke.

3.3 |. Testing the new theory at the single molecule level

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 Pi 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 Pi-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 Pi still in its active site.

In a second independent test of this hypothesis, Scott et al. (2021) determined the effect of increasing the Pi concentration to 30 mM on the single molecule mechanics and kinetics using a wild-type myosin Va construct at 100 μM ATP. If Pi-release gates the powerstroke, and therefore is released before the powerstroke, the elevated (Pi) should have reduced the amplitude of the powerstroke and/or slowed the rate the powerstroke. However, the size of the powerstroke and the rate of its development were indistinguishable from measurements made in the absence of added Pi. The duration of the longest 25% of binding events was however reduced, which is consistent with Pi rebinding to actomyosin in an ADP-bound state and accelerating detachment from actin. Similar to the observations seen with the S217A construct these findings suggest that the powerstroke is completed with Pi maintained in the active site.

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 Pi-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 Pi from its active site. Thus, these data appear to be inconsistent with the newly hypothesized mechanism where Pi-release occurs rapidly from the active site, and prior to the powerstroke, but stalls in the exit-tunnel before appearing in solution at a much slower rate (Llinas et al., 2015).

Another prediction of a powerstroke-first model is that actomyosin transiently exists in a post-powerstroke state with Pi still in the active site, but this configuration has yet to be observed in high-resolution structures. Due to its transient nature, and that such a structure requires actin, it may be difficult to capture using conventional approaches. Molecular dynamics simulations offer an alternative approach to gain insight into the transient states. Using earlier published structures, Preller and Holmes (2013) suggested that closure of the actin-binding cleft and coupled rotation of the lever arm occurred prior to release of Pi from the active site (Preller & Holmes, 2013), implying the existence of a post-powerstroke structure with Pi still in the active site, but this would need to be confirmed experimentally.

More recently, Mugnai and Thirumalai (2021) used the coordinates from Llinas et al. (2015) to simulate Pi-release. They found that the Mg++ associated with ADP needed to by hydrated by four water molecules before Pi was released (Mugnai & Thirumalai, 2021). Interestingly, Pi-release did not occur within the duration of their simulations, which lasted for several microseconds. This implies that Pi-release from the active site is likely slower than the estimates of the rate of the powerstroke (5,000/s) from the ultra-fast laser trap assay (Woody et al., 2019). Thus, these reports (Mugnai & Thirumalai, 2021; Preller & Holmes, 2013) provide indirect evidence that a post powerstroke state, with Pi remaining in the active site, may transiently exist in the cross-bridge cycle. Generating high-resolution actomyosin structures, possibly taking advantage of the transformative improvements in the resolution of cryo-EM (Zhang & Liu, 2018), combined with the ability to image filamentous actin with this method, could provide direct evidence of these structures.

4 |. CONCLUSIONS

Myosin’s powerstroke and release of Pi from the active site were originally thought to occur simultaneously; however, there is now evidence that they occur at different rates, igniting a vigorous debate over which events occurs first. Recent technological advances have enabled researchers to precisely determine the order of these events in solution. FRET-based studies in combination with transient kinetic experiments suggest that the powerstroke occurs first, and much faster than Pi-release. Subsequent crystallographic studies suggested that Pi-release may occur in two steps; one from the active site, which is fast, and a second from the exit tunnel that occurs at a much slower rate, a notion that could reconcile the disparate findings. However, a recent direct test of this hypothesis revealed data that were inconsistent with this idea, thus favoring a powerstroke-first model. Such a model would suggest that the reaction driving the powerstroke may be the formation of the actomyosin bond rather than the release of Pi form the active site. These findings therefore have important implications for understanding how myosins generate force and motion for the myriad of cellular processes they orchestrate, and how one might target these steps to treat pathologies involving myosin, including common forms of heart failure.

ACKNOWLEDGMENTS

This work was also supported by grants to Edward P. Debold from the American Heart Association (AHA; 18IPA34170048) and the National Institutes of Health (R01GM135923-01). The author thanks Chris Marang, Brent Scott, and Mike Woodward for extensive discussions on the topic of this manuscript, and Dr. Stephanie Jones for editting a final version of this manuscript.

Funding information

American Heart Association, Grant/Award Number: 18IPA34170048; NIH, Grant/Award Number: R01GM135923-01

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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