Lin J et al. (2014), Structural mechanism of the dynein power stroke.

ECB-ART-43368

Nat Cell Biol 2014 May 01;165:479-85. doi: 10.1038/ncb2939.

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Structural mechanism of the dynein power stroke.

Lin J , Okada K , Raytchev M , Smith MC , Nicastro D .

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Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. 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 domains. 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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Genes referenced: dnah3 LOC100887844 LOC100893907 LOC105444942 LOC115925415

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	Figure 2. In situ structures of sea urchin axonemal dyneins in the post-powerstroke state as revealed by cryo-ET. (a) Tomographic slice of an averaged axonemal 96 nm repeat, viewed in cross-section from the proximal side, showing the in situ arrangement of axonemal dyneins. Coloured lines indicate locations of tomographic slices shown in b, c, e, and f. (b-g) Longitudinal tomographic slices of averaged axonemal dyneins. d/dâ€™ and g/gâ€™ are magnifications of c and f. In dâ€™ and gâ€™, isosurface-rendered 3D images of averaged Î±-ODA and IDA dynein c are superimposed onto corresponding tomographic slices in d and g. (h-j) 3D isosurface renderings of averaged axonemal dyneins. 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 (b, c, e). Other labels: doublet microtubule (DMT), A-tubule (At), B-tubule (Bt), microtubule binding domain (MTBD), nexin-dynein regulatory complex (N-DRC), radial spoke (RS), microtubule polarity (+ and - end). Structure-colour coding is preserved in isosurface renderings throughout all figures. Scale bars: 10 nm.
	Figure 3. In situ structural changes of ODAs between post- and pre-powerstroke states. (a-f) Longitudinal tomographic slices of averaged axonemal dyneins in post- (a-c), pre-I (d, e) and pre-II (f) powerstroke states; note the difference in curvature of the stalks (orange arrowheads). Coloured dots and rings in a-f indicate interaction sites of the linker with specific AAA domains of the dynein heads: AAA1 (dark blue), AAA2 (light blue), AAA4 (yellow); in b and e linker and tail are indicated with magenta and pink dots, respectively. (g-j) 3D isosurface renderings of DMT in cross-view (g, i) and the conformational changes of the ODAs between post-powerstroke (h) and pre-powerstroke states (j). Coloured lines (g, i) indicate the orientation of the tomographic slices (a, c, d, f); protofilaments numbered according to Linck and Stephens31. Interactions between the linker and head are shown by docking the crystal structure of S. cerevisiae cytoplasmic dynein (ribbon representation with AAA1-6 from PDB 4AKI)12 into our EM volume (insets in h, j); note the linker base that connects to AAA1. Other labels: A-tubule (At), B-tubule (Bt), doublet microtubule (DMT), nexin-dynein regulatory complex (N-DRC). Scale bar: 10 nm.
	Figure 4. In situ structural changes of IDAs between post- and pre-powerstroke states. (a-d) 3D isosurface renderings of averaged IDAs in post-powerstroke (a, b) and pre-powerstroke states (c, d). Insets show the different curvature of the stalks (orange arrowheads) in the tomographic slices of IDA a. 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 volumes, illustrating 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, AAA1 and 2 in pre-powerstroke states. Scale bar: 10 nm (inset a, b).
	Figure 5. Schematic model of dynein movement. (a-d) Schematics of dynein in post-(D) and pre-powerstroke (D) conformations observed by cryo-ET; the interpretation of the nucleotide state of the conformations is based on previous kinetic studies3-5. Underlined dynein states (D, D-ADP, D-ADP) are predicted to have strong microtubule affinity. For a detailed explanation, see text. Other labels: microtubule polarity (+ and - end), microtubule (MT), red lines: location before step, green lines: location after one step.

References [+] :

Bouchard, erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities. 1981, Pubmed, Echinobase