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An actin-dependent conformational change in myosin

Nature Structural & Molecular Biology volume 10, pages 402–408 (2003)Cite this article

Abstract

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 Å change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23° swing of the light-chain domain.

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Figure 1: Position of probes and predicted Cα distances from cryo-EM docking of crystal structures for acto-S1 (S1 = single-headed subfragment 1 of myosin) ± ADP and transition state (S1–vanadate) with and without a 'kink' in lever arm.
Figure 2: Time-delayed LRET spectrum of myosin as a function of nucleotide and actin binding.
Figure 3: Terbium lifetime (using fluorescence at 546 nm) as a function of bound ligand.
Figure 4: Sensitized emission lifetime (measured at 570 nm) as a function of bound ligand.
Figure 5: Steady-state FRET data.

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Acknowledgements

This work was supported by NIH grants to P.R.S. and H.L.S.

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Author notes

  1. Paul R. Selvin

    Present address: Center for Biophysics and Computational Biology, University of Illinois, Urbana, 61801, Illinois, USA

  2. Ming Xiao

    Present address: Cardiovascular Research Institute, University of California, San Francisco, California, 94143, USA

Authors and Affiliations

  1. Department of Physics, University of Illinois, 1110 West Green Street, Urbana, 61801, Illinois, USA

    Ming Xiao, Jeff G. Reifenberger, Pinghua Ge & Paul R. Selvin

  2. Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, 19104-6085, Pennsylvania, USA

    Amber L. Wells, Corry Baldacchino, Li-Qiong Chen & H. Lee Sweeney

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Correspondence to H. Lee Sweeney or Paul R. Selvin.

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Xiao, M., Reifenberger, J., Wells, A. et al. An actin-dependent conformational change in myosin. Nat Struct Mol Biol 10, 402–408 (2003). https://doi.org/10.1038/nsb916

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