title: Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster.
abstract: Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.
authors: *Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA; and The Cell Adhesion Laboratory, Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, USA.
authors: Sarah M
authors: SM
authors: Heissler
authors: *Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA; and The Cell Adhesion Laboratory, Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, USA.
authors: Krishna
authors: K
authors: Chinthalapudi
authors: *Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA; and The Cell Adhesion Laboratory, Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, USA sellersj@nhlbi.nih.gov.
authors: James R
authors: JR
authors: Sellers
mesh_terms: D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D006576:Heterocyclic Compounds, 4 or More Rings; D011994:Recombinant Proteins; C472645:blebbistatin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024462:Myosin Type II
keywords: actin; blebbistatin; cytoskeleton; rotamer; transient kinetics
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