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Long single α-helical tail domains bridge the gap between structure and function of myosin VI

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Abstract

Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take ∼36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single α-helix of ∼10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the ∼36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

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Figure 1: M6 tail domains and experimental constructs.
Figure 2: CD spectra for the PT and MT-DT domains.
Figure 3: SAXS envelope reconstructions of tail domains.
Figure 4: Motility assays for the MT locked mutant compared to control M6 dimer.
Figure 5: SAXS envelope and models for full-length myosin VI.
Figure 6: A scale model of a M6 dimer moving along an actin filament.

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Acknowledgements

We thank A. Dunn, Z. Bryant and N. Geething of Stanford University for technical help with protein purification and motility assays, critical discussions and manuscript review; H.L. Sweeney of the University of Pennsylvania for plasmids; K. Holmes of the Max Planck Institute for Medical Research, Heidelberg, for the acto-myosin PDB model; T. Fenn of Stanford University for technical help with MALS analysis and graphical presentation; T. Purcell of the University of California, San Fransisco, for help with model building; S. Seifert of the Advanced Photon Source; R. Fenn of Stanford University for help with SAXS data collection; and S. Patel of Stanford University for MALDI analysis. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. B.J.S. is partially supported by grant T32 GM008294; S.D. is supported by grant PO1 GM066275; and J.A.S. is supported by grant GM33289, all from the US National Institutes of Health.

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  1. Jan Lipfert

    Present address: Present address: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, 2628 CJ Delft, The Netherlands.,

Authors and Affiliations

  1. Department of Biochemistry, Stanford University, 279 Campus Drive, Stanford, 94305, California, USA

    Benjamin J Spink, Sivaraj Sivaramakrishnan & James A Spudich

  2. Department of Physics, 382 Via Pueblo Mall, Stanford University, Stanford, 94305, California, USA

    Jan Lipfert & Sebastian Doniach

  3. Department of Applied Physics, 382 Via Pueblo Mall, Stanford University, Stanford, 94305, California, USA

    Sebastian Doniach

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Contributions

B.J.S. designed constructs, purified proteins, collected CD, MALS, DLS, gel filtration, motility and trap data, and built models; S.S. purified proteins, collected motility and trap data, and built models; J.L. collected and analyzed SAXS data; S.D. and J.A.S. helped in study design and data interpretation; all authors discussed the results and offered revisions on the manuscript.

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Correspondence to James A Spudich.

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Spink, B., Sivaramakrishnan, S., Lipfert, J. et al. Long single α-helical tail domains bridge the gap between structure and function of myosin VI. Nat Struct Mol Biol 15, 591–597 (2008). https://doi.org/10.1038/nsmb.1429

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