Edward P. Debold

Myosins are a highly conserved super family of motor proteins that are responsible for driving a host of intracellular processes in eukaryotes, from muscle contraction to vesicular transport. Myosins can perform these tasks because they transduce chemical energy, from the hydrolysis of ATP into mechanical work, in the form of a power stroke. The key event in the transduction process is the putative coupling of P _i release with the power stroke; however, the timing and mechanism of coupling of these events remain unclear. Atomic structures of myosin, captured in intermediate states of its cross-bridge cycle, suggest that P _i release is required for the power stroke to occur and therefore must precede the power stroke. In contrast, most functional assays, which can measure myosin’s structural dynamics with sub-millisecond temporal and nanometer spatial resolution, suggest that the power stroke occurs less than 1 ms after forming a strong bond with actin, while P _i release occurs 10–200 ms after binding to actin, suggesting that the power stroke precedes P _i release. A host of new studies and a few new models have been put forth in recent years to attempt to reconcile these seemingly conflicting findings. Although there is not yet a consensus on the order of these events, the new information provided by these efforts is transforming our understanding of how myosin transduces energy. This knowledge has important implications for elucidating the molecular basis of a myriad of myosin-associated diseases and, therefore, for the development of compounds to treat these diseases.

Intensely contracting fast skeletal muscle rapidly loses the ability to generate force, due in part to the accumulation of phosphate (P _i ) inhibiting myosin’s force-generating capacity, in a process that is strain dependent. Crucial aspects of the mechanism underlying this inhibition remain unclear. Therefore, we directly determined the effects of increasing [P _i ] on rabbit psoas muscle myosin’s ability to generate force against progressively higher resistive loads in a laser trap assay, with the requisite spatial and temporal resolution to discern the mechanism of inhibition. Myosin’s force-generating capacity decreased with increasing [P _i ], an effect that became more pronounced at higher resistive loads. The decrease in force resulted from myosin’s accelerated detachment from actin, which also increased at higher resistive forces. These data are well fit by a cross-bridge model in which P _i rebinds to actomyosin in a postpowerstroke, ADP-bound state before accelerating myosin’s detachment from actin. Thus, these findings provide important molecular insight into the mechanism underlying the P _i -induced loss of force during muscle fatigue from intense contractile activity.

Skeletal muscle fatigue is caused, in part, by increased production of phosphate and hydrogen ions, resulting in decreased force generation. We found that reduced force in fibers from older adults was due to altered function of myosin and actin, including slower protein interactions and reduced force per myosin head. Additionally, an ATP analog, dATP, partially reversed contractile dysfunction induced by increased phosphate and hydrogen, improving force production and altering myosin-actin interactions dependent upon fiber type.

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca²⁺ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin’s ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H⁺) act to directly and indirectly (via decreased Ca²⁺ activation) inhibit myosin’s force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases.

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (P_i) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated P_i on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of P_i-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM P_i, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of P_i from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the P_i-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of P_i-rebinding.

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 (P_i) 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 P_i 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 P_i-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 P_i, a finding most consistent with the powerstroke preceding P_i-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of P_i, indicating that the higher resistive force accelerated the rate of P_i-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k₁ = k_o 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 P_i-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 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 (P_i) 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 P_i is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that P_i‐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 P_i 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.

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_i‐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 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. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of P_i from the active site.

Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9-12, which can be activated to form functional filaments as needed13-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.