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Mechanochemical coupling of two substeps in a single myosin V motor

Nature Structural & Molecular Biology volume 11, pages 877–883 (2004)Cite this article

Abstract

Myosin V is a double-headed processive molecular motor that moves along an actin filament by taking 36-nm steps. Using optical trapping nanometry with high spatiotemporal resolution, we discovered that there are two possible pathways for the 36-nm steps, one with 12- and 24-nm substeps, in this order, and the other without substeps. Based on the analyses of effects of ATP, ADP and 2,3-butanedione 2-monoxime (a reagent shown here to slow ADP release from actomyosin V) on the dwell time and the occurrence frequency of the main and the intermediate states, we propose that the 12-nm substep occurs after ATP binding to the bound trailing head and the 24-nm substep results from a mechanical step following the isomerization of an actomyosin-ADP state on the bound leading head. When the isomerization precedes the 12-nm substep, the 36-nm step occurs without substeps.

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Figure 1: Stepwise movement of single myosin V motor under various external loads.
Figure 2: The time course of myosin V movement at a 10-kHz sampling rate and force dependence of the occurrence of various steps.
Figure 3: Force dependence of the intermediate and the main dwell times at various nucleotide states and of the occurrence frequency of the intermediate state.
Figure 4: Effects of BDM on ATPase cycle kinetics of single-headed myosin V.
Figure 5: Hand-over-hand model coupled with nucleotide states explaining the present results.

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Acknowledgements

We thank M.R. Webb for the phosphate-binding protein clone and suggestions on purification and labeling, and H.L. Sweeney for providing the myosin V heavy and light chain viruses. We are grateful to N. Sasaki, M.Y. Ali, K. Kinosita, Jr. and E.M. Ostap for encouragement and stimulating discussions. This research was partly supported by Grants-in-Aid for Specially Promoted Research, for the Bio-venture Project and for The 21st Century COE Program (Physics of Self-Organization Systems) at Waseda Univ. from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to S.I.) and supported by a Scientist Development Grant from the American Heart Association and a grant from the US National Science Foundation (to E.M.D.L.C.). S.U. is a postdoctoral fellow of the Japan Society for the Promotion of Science. A.O.O. is supported by a Cellular & Molecular Biology graduate training grant (Yale University).

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Authors and Affiliations

  1. Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Sotaro Uemura & Shin'ichi Ishiwata

  2. Department of Metallurgy, Graduate School of Engineering, Tohoku University, Sendai, 980-8579, Japan

    Hideo Higuchi

  3. Center of Interdisciplinary Research, Tohoku University, Sendai, 980-8579, Japan

    Hideo Higuchi

  4. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06520, Connecticut, USA

    Adrian O Olivares & Enrique M De La Cruz

  5. Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan

    Shin'ichi Ishiwata

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Correspondence to Shin'ichi Ishiwata.

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Uemura, S., Higuchi, H., Olivares, A. et al. Mechanochemical coupling of two substeps in a single myosin V motor. Nat Struct Mol Biol 11, 877–883 (2004). https://doi.org/10.1038/nsmb806

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