Direct detection of the myosin super-relaxed state and interacting-heads motif in solution

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Abstract

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the forcegenerating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.

Original languageEnglish (US)
Article number101157
JournalJournal of Biological Chemistry
Volume297
Issue number4
Early online dateSep 2 2021
DOIs
StatePublished - Oct 1 2021

Bibliographical note

Funding Information:
Funding and additional information—This work was supported by NIH grants to D. D. T. (R01 AR032961, R37 AG26160). S. C. was supported by NIH training grant to D. D. T. (T32 AR007612). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

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