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Structural mechanism of the dynein power stroke

Abstract

Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility1,2. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains3,4,5. However, key structural details of dynein’s force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.

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Figure 1: Dynein and its arrangement in sea urchin sperm flagella.
Figure 2: In situ structures of sea urchin axonemal dyneins in the post-power-stroke state as revealed by cryo-electron tomography.
Figure 3: In situ structural changes of ODAs between post- and pre-power-stroke states.
Figure 4: In situ structural changes of IDAs between post- and pre-power-stroke states.
Figure 5: Schematic model of dynein movement.

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Acknowledgements

We thank D. T. N. Chen (Brandeis University) for providing Strongylocentrotus purpuratus sperm; M. Porter (University of Minnesota) for providing the pseudo wild-type Chlamydomonas strain; and C. Xu for providing training and management of the Brandeis EM facility. We are grateful to D. Mitchell (SUNY Upstate Medical University), and J. Gelles, D. DeRosier and T. Heuser (all from Brandeis University) for critically reading the manuscript. This work was supported by financial support from the National Institutes of Health (GM083122 to D.N.).

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Authors

Contributions

D.N. conceived and directed the study. J.L. performed the experiments. J.L., M.R. and D.N. analysed the data. J.L., K.O., M.C.S. and D.N. wrote the manuscript.

Corresponding author

Correspondence to Daniela Nicastro.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distributions of different dynein conformations inside a sea urchin sperm flagellum revealed by classification analysis.

Subtomogram volumes containing the 96 nm axonemal repeat were aligned and classified, focusing on β-ODA. The distributions of the different classified conformations were then mapped back to their locations in the raw tomograms of the sea urchin sperm flagella. Different conformations of β-ODA (and the o-SUB5-6 bridge structures) are indicated by different coloured dots: post-powerstroke (red), pre-II (blue), minor and low resolution classes possibly representing other dynein conformations (orange), o-SUB5-6 bridge structure (green); note that the distribution of identified dynein conformations is highly specific to particular doublets, which is consistent with the precise regulation of dynein activity to generate the flagellar waveform.

Supplementary Figure 2 In situ structures of Chlamydomonas axonemal dyneins in post-powerstroke state as revealed by cryo-ET.

(a) Tomographic slice of an averaged doublet microtubule (DMT) viewed in cross-section from proximal, showing the in situ arrangement of the axonemal dyneins. Coloured lines indicate locations of tomographic slices in bd and f,g. (bh’) Longitudinal (parallel to the microtubule) tomographic slices show the averaged axonemal dyneins. e/e’ and h/h’ are zoom-ins from d and g, respectively. In e’ and h’, isosurface-rendered 3D images of averaged α-ODA and IDA dynein c were superimposed on the corresponding tomographic slices (e, h). (ik) 3D isosurface renderings show the averaged axonemal dyneins. Note that each Chlamydomonas ODA is composed of three dynein heavy chains (α, β, and γ-ODA); the innermost γ-ODA corresponds to the sea urchin α-ODA (compare to Fig. 2). Tail (pink), linker (magenta) and head (green) domains are clearly visible in both tomographic slices and isosurface renderings; coiled-coil stalks (orange arrowheads) are distinct in the tomographic slices (bd, f). IDA a, b, c, e, g, d are also known as IDA2, x,3,4,5,6, respectively36. Other labels: A-tubule (At), B-tubule (Bt), nexin-dynein regulatory complex (N-DRC), radial spoke (RS), radial spoke 3 stand-in (RS3S). Structure-colour coding is preserved in the isosurface renderings in all following figures. White arrowheads in e, e’, and j highlight an extra density that is specifically attached to the dynein head and tail domains. Scale bars: 10 nm.

Supplementary Figure 3 In situ structural changes of sea urchin IDAs between post- and pre-powerstroke states.

(ad) 3D isosurface rendering of averaged IDAs showing conformational changes observed for IDA dyneins c and e between post-powerstroke (a, b) and pre-powerstroke (c, d) states. In b and d, the crystal structure of S. cerevisiae cytoplasmic dynein (ribbon representation with AAA1-6 from PDB 4AKI)12 is docked into our IDA EM volume to illustrate changes in the interaction between the linker and head. White arrowheads in c and d highlight an extra density that specifically attaches to the linker and AAA1 in pre-powerstroke states.

Supplementary Figure 4 Comparison between simulated and real 3D structures of dynein in different conformational states.

The averaged 3D cryo-ET structures of two different dynein isoforms (IDA dynein a and β-ODA) in two different conformational states (post- and pre-II-powerstroke states) provide structural details of the relative motion between the AAA-domain head and the linker. The first column on the left shows the x-ray crystal structure of S. cerevisiae cytoplasmic dynein (PDB 4AKI A chain)12. To interpret the relative motions between different conformational states, the crystal structure was cut at the proposed hinge site (arrow heads); the major part of the linker was then aligned (held stationary), while the dynein head and stalk were rotated around the hinge site to the locations predicted by our different cryo-ET structures; the rotation angle between the head and linker increases from top to bottom: IDA a, post-powerstroke (top); β-ODA, post-powerstroke; IDA a, pre-II state; β-ODA; pre-II state (bottom). The second column shows simulated density maps that were generated from the modified x-ray structures using the Chimera molmap command. The third column shows the 3D in situ structures of sea urchin axonemal dyneins (with the major part of their linker aligned) as revealed by cryo-ET. The fourth column shows the same structures as the third, but with the crystal structures from the first column docked into the cryo-ET structures. The linker and tail domains in the cryo-ET structures were coloured in magenta and pink, respectively.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1094 kb)

Live cell DIC light microscopy video of beating Strongylocentrotus (sea urchin) sperm.

This video shows sea urchin sperm, rapidly swimming due to actively beating flagella. In this preparation, freshly harvested sea urchin sperm were diluted in artificial seawater. (AVI 14731 kb)

Live cell DIC light microscopy video of inhibited Strongylocentrotus (sea urchin) sperm.

This video shows immotile sea urchin sperm. In this preparation, freshly harvested sea urchin sperm were diluted in artificial seawater containing the ATPase (dynein) inhibitor EHNA (2 mM). Visible movement of the sea urchin sperm is passive, due to buffer flow. (AVI 12182 kb)

Mechanistic model of dynein movement.

This video combines real electron microscopic data and a schematic model of the powerstroke cycle of dynein. The first part of the video shows three representative tomographic slices of dynein in the post-, pre-I and pre-II powerstroke states. Initially, a schematic outline of dynein is superimposed on each of these slices to highlight conformational changes of dynein domains. The second part of the video highlights the proposed head-rotation mechanism of dynein movement, as described in this study. (AVI 17612 kb)

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Lin, J., Okada, K., Raytchev, M. et al. Structural mechanism of the dynein power stroke. Nat Cell Biol 16, 479–485 (2014). https://doi.org/10.1038/ncb2939

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