Myosin cleft movement and its coupling to actomyosin dissociation

Abstract

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.

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Figure 1: Models to show the location of Dd residues 416 (cyan) and 537 (yellow) that are labeled with pyrene.
Figure 2: Fluorescence emission spectra of pyr-Md (365 nm excitation).
Figure 3: Time-resolved pyrene fluorescence measurements.
Figure 4: Stopped-flow records of pyrene excimer fluorescence on interaction of pyr-Md with actin and nucleotides.

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Acknowledgements

We thank W. Shih and J. Spudich for the cysteine-deficient construct and K. Holmes and R. Schroeder for the coordinates of their actomyosin model. We are grateful to the Wellcome Trust, the UK Biotechnology and Biological Sciences Research Council, the US National Science Foundation and the Magyary Zoltán Foundation for financial support.

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Correspondence to Clive R Bagshaw.

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The authors declare no competing financial interests.

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Conibear, P., Bagshaw, C., Fajer, P. et al. Myosin cleft movement and its coupling to actomyosin dissociation. Nat Struct Mol Biol 10, 831–835 (2003). https://doi.org/10.1038/nsb986

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