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Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Nature volume 442, pages 208–211 (2006)Cite this article

Abstract

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking1. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can ‘walk’ hand-over-hand along an actin filament for many steps before detaching2,3,4. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5–7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact8,9,10. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head–S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

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Figure 1: Electron micrographs and averages of myoV.
Figure 2: Molecular arrangement within the ‘flower’ motif.
Figure 3: Placement of the cargo-binding domain on the motor domain.
Figure 4: Orientation of the myoV-inhibited structure on F-actin. F-actin strands rendered light blue and grey.

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References

  1. Reck-Peterson, S. L., Provance, D. W. Jr, Mooseker, M. S. & Mercer, J. A. Class V myosins. Biochim. Biophys. Acta 1496, 36–51 (2000)

    Article  CAS  Google Scholar 

  2. Mehta, A. D. et al. Myosin-V is a processive actin-based motor. Nature 400, 590–593 (1999)

    Article  ADS  CAS  Google Scholar 

  3. Forkey, J. N., Quinlan, M. E., Shaw, M. A., Corrie, J. E. & Goldman, Y. E. Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature 422, 399–404 (2003)

    Article  ADS  CAS  Google Scholar 

  4. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003)

    Article  ADS  CAS  Google Scholar 

  5. Wang, F. et al. Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V. J. Biol. Chem. 275, 4329–4335 (2000)

    Article  CAS  Google Scholar 

  6. Homma, K., Saito, J., Ikebe, R. & Ikebe, M. Ca2+-dependent regulation of the motor activity of myosin V. J. Biol. Chem. 275, 34766–34771 (2000)

    Article  CAS  Google Scholar 

  7. Trybus, K. M., Krementsova, E. & Freyzon, Y. Kinetic characterization of a monomeric unconventional myosin V construct. J. Biol. Chem. 274, 27448–27456 (1999)

    Article  CAS  Google Scholar 

  8. Wang, F. et al. Regulated conformation of myosin V. J. Biol. Chem. 279, 2333–2336 (2004)

    Article  CAS  Google Scholar 

  9. Krementsov, D. N., Krementsova, E. B. & Trybus, K. M. Myosin V: Regulation by calcium, calmodulin, and the tail domain. J. Cell Biol. 164, 877–886 (2004)

    Article  CAS  Google Scholar 

  10. Li, X. D., Mabuchi, K., Ikebe, R. & Ikebe, M. Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change. Biochem. Biophys. Res. Commun. 315, 538–545 (2004)

    Article  CAS  Google Scholar 

  11. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies (Academic, San Diego, CA, 1996)

    Google Scholar 

  12. Terrak, M., Wu, G., Stafford, W. F., Lu, R. C. & Dominguez, R. Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs-functional implications. EMBO J. 22, 362–371 (2003)

    Article  CAS  Google Scholar 

  13. Coureux, P. D., Sweeney, H. L. & Houdusse, A. Three myosin V structures delineate essential features of chemo-mechanical transduction. EMBO J. 23, 4527–4537 (2004)

    Article  CAS  Google Scholar 

  14. Tama, F., Miyashita, O. & Brooks, C. L. Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM. J. Struct. Biol. 147, 315–326 (2004)

    Article  CAS  Google Scholar 

  15. Cheney, R. E. et al. Brain myosin-V is a two-headed unconventional myosin with motor activity. Cell 75, 13–23 (1993)

    Article  CAS  Google Scholar 

  16. Sweeney, H. L. et al. Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket. J. Biol. Chem. 273, 6262–6270 (1998)

    Article  CAS  Google Scholar 

  17. Sellers, J. R. Regulation of cytoplasmic and smooth muscle myosin. Curr. Opin. Cell Biol. 3, 98–104 (1991)

    Article  CAS  Google Scholar 

  18. Hackney, D. D., Levitt, J. D. & Suhan, J. Kinesin undergoes a 9 S to 6 S conformational transition. J. Biol. Chem. 267, 8696–8701 (1992)

    CAS  PubMed  Google Scholar 

  19. Wendt, T., Taylor, D., Trybus, K. M. & Taylor, K. Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2. Proc. Natl Acad. Sci. USA 98, 4361–4366 (2001)

    Article  ADS  CAS  Google Scholar 

  20. Stock, M. F. et al. Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity. J. Biol. Chem. 274, 14617–14623 (1999)

    Article  CAS  Google Scholar 

  21. Coy, D. L., Hancock, W. O., Wagenbach, M. & Howard, J. Kinesin's tail domain is an inhibitory regulator of the motor domain. Nature Cell Biol. 1, 288–292 (1999)

    Article  CAS  Google Scholar 

  22. Hackney, D. D. & Stock, M. F. Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release. Nature Cell Biol. 2, 257–260 (2000)

    Article  CAS  Google Scholar 

  23. Wang, F. S., Wolenski, J. S., Cheney, R. E., Mooseker, M. S. & Jay, D. G. Function of myosin-V in filopodial extension of neuronal growth cones. Science 273, 660–663 (1996)

    Article  ADS  CAS  Google Scholar 

  24. Espreafico, E. M. et al. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J. Cell Biol. 119, 1541–1557 (1992)

    Article  CAS  Google Scholar 

  25. Star, E. N., Kwiatkowski, D. J. & Murthy, V. N. Rapid turnover of actin in dendritic spines and its regulation by activity. Nature Neurosci. 5, 239–246 (2002)

    Article  CAS  Google Scholar 

  26. Forscher, P. & Smith, S. J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J. Cell Biol. 107, 1505–1516 (1988)

    Article  CAS  Google Scholar 

  27. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    Article  CAS  Google Scholar 

  28. Winkler, H. & Taylor, K. A. Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy 106, 240–254 (2006)

    Article  CAS  Google Scholar 

  29. Winkler, H. & Taylor, K. A. Focus gradient correction applied to tilt series image data used in electron tomography. J. Struct. Biol. 143, 24–32 (2003)

    Article  Google Scholar 

  30. Rayment, I. et al. Structure of the actin–myosin complex and its implications for muscle contraction. Science 261, 58–65 (1993)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We thank H. Winkler for his guidance in focus gradient correction and 3D volume classification. We also thank the National Institutes of Health Research Resource for MMTSB and C. L. Brooks 3rd for making NMFF available. This research was supported by grants from the National Institutes of Health (to K.M.T. and K.A.T.).

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Authors and Affiliations

  1. The Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, 32306-4380, USA

    Jun Liu, Dianne W. Taylor & Kenneth A. Taylor

  2. Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont, 05405-0068, USA

    Elena B. Krementsova & Kathleen M. Trybus

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Corresponding authors

Correspondence to Kathleen M. Trybus or Kenneth A. Taylor.

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Competing interests

The atomic coordinates of the final myoV atomic model have been deposited in the Protein Data Bank with accession number 2DFS. The density volume for the flower motif shown in Fig. 2 has been deposited in the European Bioinformatics Institute under accession code EMD-1201. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Movie Legends and Supplementary Figure Legends. This file also contains Supplementary Methods (model fitting, Electron micrographs of actin decorated with myosin V in the inhibited conformation and a description of the conditions used to make the specimens of myoV decorated actin. (PDF 689 kb)

Supplementary Figures

Contains a slide show presentation showing the initial models, initial dockings and final results of flexible fitting (PPT 2129 kb)

Supplementary Movie 1

Shows a tilt series of one of the myosin V 2-D arrays and an image of the tomogram obtained from it. (MOV 8635 kb)

Supplementary Movie 2

Shows the flower motif density envelope and the fitted atomic model with rotation and different degrees of zoom. (MOV 9217 kb)

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Liu, J., Taylor, D., Krementsova, E. et al. Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature 442, 208–211 (2006). https://doi.org/10.1038/nature04719

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