Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin.

Autor:	Tang W; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania., Ge J; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania., Unrath WC; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania., Desetty R; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania., Yengo CM; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania. Electronic address: cmy11@psu.edu.
Jazyk:	angličtina
Zdroj:	Biophysical journal [Biophys J] 2021 Jun 01; Vol. 120 (11), pp. 2222-2236. Date of Electronic Publication: 2021 Apr 20.
DOI:	10.1016/j.bpj.2021.04.007
Abstrakt:	Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction. (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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