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J Biol Chem. 2019 Feb 1;294(5):1554-1567. doi: 10.1074/jbc.RA118.006128. Epub 2018 Dec 5.

Converter domain mutations in myosin alter structural kinetics and motor function.

Author information

1
From the Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033 and.
2
the Department of Biochemistry, Biophysics and Molecular Biology, University of Minnesota, Minneapolis, Minnesota 55455.
3
From the Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033 and cmy11@psu.edu.

Abstract

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

KEYWORDS:

ATPase; actin; cardiomyopathy; fluorescence resonance energy transfer (FRET); mechanochemistry; motor protein; muscle; myosin; structural kinetics

PMID:
30518549
PMCID:
PMC6364761
[Available on 2020-02-01]
DOI:
10.1074/jbc.RA118.006128

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