Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

• Published in: The Journal of biological chemistry Vol. 288; no. 39; pp. 28312 - 28323
• Main Authors: Cochran, Jared C., Thompson, Morgan E., Kull, F. Jon
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.09.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Actin; Actin Cytoskeleton; Actins - chemistry; Adenosine Triphosphatases - chemistry; Adenosine Triphosphate - chemistry; Allosteric Site; ATPases; Cysteine - chemistry; Dictyostelium - enzymology; Dictyostelium - genetics; Gene Expression Regulation; Hydrolysis; Magnesium - chemistry; Manganese; Manganese - chemistry; Metals - chemistry; Molecular Biophysics; Molecular Motors; Myosin; Myosin Type II - genetics; Myosin Type II - metabolism; Nucleotides - chemistry; Protein Binding; Protein Structure, Tertiary; Serine - chemistry; Molecular Motors; ATPases; Actin; Myosin; Manganese
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.466045

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg2+) 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 Mg2+- or Mn2+-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn2+, 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. Mn2+ 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. Background: Myosins convert the energy of ATP hydrolysis into force production. Results: Substitution of the metal-interacting serine in switch-1 with cysteine renders the motor sensitive to manganese. Conclusion: This technology provides a reversible structural linkage between the nucleotide pocket and actin-binding region in the myosin motor domain. Significance: Understanding the ATPase mechanism requires a description of allosteric communication in molecular motors.