Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments.
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.
['Cochran|Jared C|JC|', 'Thompson|Morgan E|ME|', 'Kull|F Jon|FJ|']
D008841:Actin Cytoskeleton D000199:Actins D000251:Adenosine Triphosphatases D000255:Adenosine Triphosphate D000495:Allosteric Site D003545:Cysteine D004023:Dictyostelium D005786:Gene Expression Regulation D006868:Hydrolysis D008274:Magnesium D008345:Manganese D008670:Metals D024462:Myosin Type II D009711:Nucleotides D011485:Protein Binding D017434:Protein Structure, Tertiary D012694:Serine
Myosin Actin ATPases Manganese Molecular Motors