Converter domain mutations in myosin alter structural kinetics and motor function

Laura K. Gunther, John A. Rohde, Wanjian Tang, Shane D. Walton, William C. Unrath, Darshan V. Trivedi, Joseph M. Muretta, David D. Thomas, Christopher M. Yengo

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1554-1567
Number of pages14
JournalJournal of Biological Chemistry
Volume294
Issue number5
DOIs
StatePublished - Feb 1 2019

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health Grant HL127699 (to C. M. Y.) and Grants R01AR32961 and R37AG26160 (to D. D. T.) and Ameri-can Heart Association Grant 14SDG20480032 (to J. M. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Tables S1 and S2 and Figs. S1–S8. 1 These authors contributed equally to this work. 2Supported by National Research Service Award Postdoctoral Fellowship F32DC016788. 3 To whom correspondence should be addressed. Tel.: 717-531-8575; E-mail: cmy11@psu.edu.

Publisher Copyright:
© 2019 Gunther et al.

Keywords

  • Adenosine Diphosphate/metabolism
  • Adenosine Triphosphatases/metabolism
  • Adenosine Triphosphate/metabolism
  • Amino Acid Sequence
  • Animals
  • Chickens
  • Mechanotransduction, Cellular
  • Models, Molecular
  • Motor Activity
  • Mutation
  • Myosin Type V/chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Sequence Homology

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article
  • Research Support, N.I.H., Extramural

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