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Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Nature Structural & Molecular Biology volume 24, pages 461468 (2017) | Download Citation

Abstract

Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor–motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.

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Acknowledgements

We thank C. Moores, G. Zanetti and A. Osborne for critical comments on the manuscript; M. Williams, S. Reck-Peterson and members of the Birkbeck EM group for advice; S. Nofal, S. Miah, and L. Stejskal for initial experiments; and A. Carter (MRC-LMB, Cambridge) for plasmids. This work was supported by a Sir Henry Dale Fellowship to A.J.R. from the Wellcome Trust and Royal Society [104196/Z/14/Z].

Author information

Affiliations

  1. Institute of Structural and Molecular Biology, Birkbeck, London, UK.

    • Katerina Toropova
    • , Miroslav Mladenov
    •  & Anthony J Roberts

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Contributions

K.T. and A.J.R. conducted biochemical and TIRF experiments. K.T. performed electron microscopy experiments. M.M., K.T., and A.J.R. generated and purified constructs. K.T. and A.J.R. analyzed data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Anthony J Roberts.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6

Text files

  1. 1.

    Supplementary Data : Model of dynein-2 in its autoinhibited state.

    Coordinates for the linker-stacking model of dynein-2 autoinhibition, derived from PDB 4RH7.

Videos

  1. 1.

    Impact of motor concentration on dynein-2 microtubule gliding.

    Microtubule gliding powered by Dyn2motor at different concentrations. See also Fig. 2d. Videos are shown at 24X real time.

  2. 2.

    Dynein-2 dimer architecture in different nucleotide conditions.

    Class averages of GST-Dyn2motor in no nucleotide and 1 mM ATP conditions. See also Fig. 4b. Videos show 38 class averages in each condition, looped 4 times.

  3. 3.

    Pairs of dynein-2 motor domains in crystallo match the architecture of isolated dimers.

    Analysis of PDB 4RH7 crystal lattice reveals pairs of monomeric dynein-2 motor domains matching the stacked architecture of Dyn2motor dimers observed by single-particle electron microscopy.

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https://doi.org/10.1038/nsmb.3391

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