Mechanism of Blebbistatin Inhibition of Myosin II

Abstract

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...

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

“…Therefore, we think that the inhibition of myosin II motor activity by the phosphorylation of MLC 20 at the inhibitory sites is in part responsible for the PDGF-induced disassembly of stress fibers and the decrease in the focal adhesion. Supporting this view, blebbistatin, a specific inhibitor of actin-activated myosin II ATPase activity but not the filament formation, blocked the formation of actomyosin stress fibers and focal adhesions (Straight et al, 2003;Kovacs et al, 2004;Hotulainen and Lappalainen, 2006). This result suggests that the myosin motor activity is critical for the formation of stress fibers and focal adhesions.…”

Material‐Driven Fibronectin Assembly Promotes Maintenance of Mesenchymal Stem Cell Phenotypes

“…We used Y-27632 as specifi c inhibitor of Rho-kinase, and blebbistatin as specifi c inhibitor of myosin II activity. [ 37,38 ] Staining for pMLC was reduced in a dose-dependent manner (10 × 10 −6 and 20 × 10 −6 M , respectively) in the presence of these inhibitors in cells on the different substrates ( Figure S3, Supporting Information).…”

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

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