The structural basis of blebbistatin inhibition and specificity for myosin II

Abstract

Molecular motors play a central role in cytoskeletal-mediated cellular processes and thus present an excellent target for cellular control by pharmacological agents. Yet very few such compounds have been found. We report here the structure of blebbistatin, which inhibits specific myosin isoforms, bound to the motor domain of Dictyostelium discoideum myosin II. This reveals the structural basis for its specificity and provides insight into the development of new agents.

Inhibiting nonmuscle myosin II impedes inflammatory infiltration and ameliorates progressive renal disease

“…This effect resembled that of direct NMII suppression by 2,3-butanedione monoxime, a myosin blockade, 47 although the specificity of this chemical inhibitor is not as satisfactory as newly discovered ones such as blebbistatin. 12 Consistent with these findings, NMII was lately found to be associated with the uropod during T-lymphocyte crawling 13,14 and knockdown of NMIIA by RNA interference resulted in a defect in tail retraction in T cells and reduced its crawling activity on either ICAM-1 coated 13 or plain culture surface. 14 Besides neutrophils and T lymphocytes, NMII is also indispensable for migration and chemotaxis in macrophages and other leukocytes.…”

“…It behaves as an uncompetitive inhibitor and functions by binding the large cleft in the motor domain, which opens and closes during the contractile cycle. 11,12 Blebbistatin specifically stabilizes the metastable or transition state of myosin. 12 Binding of blebbistatin to myosin II leads to a long-lived complex of myosin with adenosine diphosphate and inorganic phosphate, which occurs before the force-generating step catalyzed by the release of phosphate on the rebinding of myosin with actin.…”

“…11,12 Blebbistatin specifically stabilizes the metastable or transition state of myosin. 12 Binding of blebbistatin to myosin II leads to a long-lived complex of myosin with adenosine diphosphate and inorganic phosphate, which occurs before the force-generating step catalyzed by the release of phosphate on the rebinding of myosin with actin. 50 Thus, blebbistatin inhibits the transition into force-producing states.…”

“…50 Thus, blebbistatin inhibits the transition into force-producing states. 12 Blebbistatin has been widely accepted and used as a highly selective blockade for NMII. Previous studies have shown that blebbistatin was able to paralyze the myosin power stroke, inhibit motility, and blunt migration and chemotaxis in a variety of cultured leukocytes, 13,14,16,18,19 suggesting that inhibition of NMII might ameliorate inflammatory infiltration.…”

“…11 It is a cell-permeable 1-phenyl-1-2-pyrrolidinone derivative that shows high affinity and selectivity toward NMII adenosine triphosphatase activity with minimal effects on activity of smooth muscle myosin II or other types of myosin. 12 Blebbistatin was recently found to mitigate the migration of a variety of immune competent cells, [13][14][15][16][17][18] suggesting that NMII is crucial for immunocyte motility. Consistently, a growing body of evidence reveals that indirect inactivation of NMII through inhibition of myosin light chain kinase 19,20 or Rho kinase 18,[21][22][23][24] impaired leukocyte migration and improved inflammation.…”