Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatinbinding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.Myosin IIs are ATP-driven molecular motors forming an essential part of the motile machinery of most eukaryotic cell types examined. Among other functions, they serve such diverse and vital functions as muscle contraction, cytokinesis, cortical tension maintenance, and neurite outgrowth and retraction (1-6). In studies of myosin II, the use of enzyme inhibitors can be a powerful approach, provided that selective and high affinity compounds are available that do not interfere with other cellular processes. The importance of the latter aspect is emphasized by a recent study (7) that showed that 2,3-butanedione-monoxime, a compound widely used to inhibit myosin II, is not specific and probably affects the function of a wide range of proteins.Blebbistatin was recently discovered as a small molecule inhibitor of muscle and non-muscle myosin II (8). The compound is permeable to cell membranes. It is a potent inhibitor of skeletal muscle and non-muscle myosin II isoforms, although it has little or no effect on smooth muscle myosin II and myosins from classes I, V, and X (9). Because of its selectivity and high affinity for several class II myosins, blebbistatin has the potential to become a popular tool in the fields of cell motility and muscle physiology. For the interpretation of the cellular effects caused by blebbistatin, a detailed understanding is essential of its effects on the myosin II ATPase and on the interaction of the myosin head with actin and substrate. We undertook an in-depth characterization of the effect of blebbistatin on the functional properties of rabbit skeletal muscle myosin II, and we performed blind docking simulations on various atomic structures of the myosin head to determine the binding site and the structural basis of isoform specificity of the inhibitor.We find that blebbistatin exerts its inhibitory effect by binding to the myosin-ADP-P i complex with hi...

Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.T he mechanical performance of myosin 2 filaments interacting with actin is strongly determined by the duty ratio, which is the fraction of the mechanical cycle time that each myosin head is strongly attached to actin. If load changes the kinetics of the ATPase cycle that underlies the mechanical cycle, then the lifetimes and steady-state abundance of actin-bound and detached intermediates will change with profound effects on function.Duty ratio varies widely in myosin 2. Skeletal muscle myosin 2 is the classical low duty ratio myosin, adapted for rapid contraction. It is able to sustain loads because it forms long, bipolar filaments containing Ϸ150 molecules in each half, so load can be sustained when only a small fraction of the heads are attached at any time. By contrast, nonmuscle myosin 2 (NM2), essential for cytokinesis, tension maintenance, and contractility of nonmuscle and smooth muscle cells (1-4), assembles into short filaments with only Ϸ10 molecules in each half (5, 6), so their duty ratios under load need to be higher to avoid slippage. The unloaded kinetic mechanisms of single-headed fragments of the NM2A and NM2B isoforms show that NM2A is the faster, lower duty ratio motor, whereas NM2B is slow, with a higher duty ratio suggesting a role in tension maintenance (7-9).Load-dependent increase of NM2 duty ratio would make the distinction from skeletal muscle myosin more extreme. It could lead to both heads of the molecule being simultaneously attached to the same actin filament as occurs in the absence of ATP, a situation believed to be rare in active skeletal muscle. Kinetic and mechanical studies on smooth muscle myosin 2 have demonstrated load dependence of its lifetime of attachment (10,11). Because ADP release is associated with axial translation in many myosins, load is expected to affect ADP release kinetics (12). Indeed, differential ADP affinities of the two heads of actin-bound smooth muscle heavy meromyosin have been indicated (13). However, there are no direct measurements of the kinetics of ADP release from mechanically loaded heads of any myosin 2 in solution...

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.