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

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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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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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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