title: Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments.
abstract: G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg(2+)) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg(2+)- or Mn(2+)-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn(2+), providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn(2+) rescues this effect to near wild-type activity. 2'(3')-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.
authors: From the Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405.
authors: Jared C
authors: JC
authors: Cochran
authors: Morgan E
authors: ME
authors: Thompson
authors: F Jon
authors: FJ
authors: Kull
mesh_terms: D008841:Actin Cytoskeleton; D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000495:Allosteric Site; D003545:Cysteine / Q000737:chemistry; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry
chemical_list: D000199:Actins; D008670:Metals; D009711:Nucleotides; D008345:Manganese; D012694:Serine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024462:Myosin Type II; D008274:Magnesium; D003545:Cysteine
keywords: ATPases; Actin; Manganese; Molecular Motors; Myosin
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