title: The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.
abstract: The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.
authors.affiliation: From the School of Biosciences, University of Kent, CT2 7NJ Canterbury, United Kingdom and.
authors.forename: Marieke J
authors.initials: MJ
authors.lastname: Bloemink
authors.affiliation: the Department of Biology, Molecular Biology Institute, and SDSU Heart Institute at San Diego State University, San Diego, California 92182-4614.
authors.forename: Girish C
authors.initials: GC
authors.lastname: Melkani
authors.affiliation: the Department of Biology, Molecular Biology Institute, and SDSU Heart Institute at San Diego State University, San Diego, California 92182-4614. Electronic address: sbernstein@mail.sdsu.edu.
authors.forename: Sanford I
authors.initials: SI
authors.lastname: Bernstein
authors.affiliation: From the School of Biosciences, University of Kent, CT2 7NJ Canterbury, United Kingdom and. Electronic address: m.a.geeves@kent.ac.uk.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Geeves
mesh_terms: D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D024744:Skeletal Muscle Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024744:Skeletal Muscle Myosins
keywords: actin; fluorescence; homology modeling; kinetics; muscle; myosin; protein structure-function; sequence alignment