Structure and functional role of dynein's microtubule-binding domain

Abstract

Dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. An unusual feature of dynein is that its microtubule-binding domain (MTBD) is separated from its ring-shaped AAA+ adenosine triphosphatase (ATPase) domain by a 15-nanometer coiled-coil stalk. We report the crystal structure of the mouse cytoplasmic dynein MTBD and a portion of the coiled coil, which supports a mechanism by which the ATPase domain and MTBD may communicate through a shift in the heptad registry of the coiled coil. Surprisingly, functional data suggest that the MTBD, and not the ATPase domain, is the main determinant of the direction of dynein motility.

Fig. 1

Crystal structure of the dynein microtubule-binding domain. (A) Cartoon of a dynein motor (heavy chain). A gray box highlights the region of the stalk and microtubule-binding domain (MTBD) whose atomic structure is reported. (B) Effect of different heptad registries on microtubule-binding affinity of monomeric SRS-MTBD fusions with stalks of one-quarter and full native length. Paired numbers designating each construct (e.g., 22:19) indicate the number of residues between the SRS splice site and the proline marking the stalk-MTBD boundary for CC1 and CC2, respectively. (C) Crystal structure of the MTBD, showing the two α helices of the stalk (CC1, purple; CC2, red) that extend out of the SRS coiled coil and connect to the six-helix bundle (H1 to H6) forming the MTBD proper. A staggered pair of conserved prolines (Pro³²⁸⁵ and Pro³⁴⁰⁹) are associated with a kink in the stalk. Inset: dimeric SRS-MTBD fusion protein (chain A, blue SRS with red MTBD; chain B, gray). (D) Schematic diagram of the stalk helices (CC1 and CC2) showing the heptad repeat hydrophobic contacts (blue lines) in the core of the coiled coil. The regularity of this repeat is disrupted between the conserved prolines (magenta), resulting in a half-heptad shift in coiled-coil registry. Residues in the SRS are shown in green. Residues in CC1 and CC2 that contact the other helices in MTBD are marked with red dots Abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

Fig. 2

Microtubule-binding surface of the dynein MTBD. (A) Close-up view of the MTBD, showing the putative microtubule-binding helices H1, H3, and H6. Residues that abolish binding when mutated to alanine (13) are shown in stick representation. The inset shows this interface (boxed) with respect to the whole MTBD structure. (B) A model of the dynein MTBD (crystal structure shown and colored as in Fig. 1C) bound to a tubulin protofilament (α tubulin, green; β tubulin, yellow). Crystal structures were docked into a single protofilament cut from the cryo-EM electron density map of a microtubule decorated with a monomeric, tight-binding (α registry) SRS-MTBD construct containing 12 heptads of dynein stalk (SRS-MTBD-85:82). The plus and minus signs indicate microtubule polarity. The SRS and nine basal heptad repeats of the stalk are not visible in the reconstructed image (gray arrows). (C) Conformational differences between the A and B MTBD monomers on an SRS dimer. Regions of high RMS difference are colored dark red. (D) Close-up of the H1–H3 interface. Aligned MTBDs are shown in light (chain B) and dark (chain A) coloring. The contact between the highly conserved Pro³³¹¹ and Trp³³³⁴ residues is broken in chain B.

Fig. 3

Rotation of the AAA+ ring by insertion or deletion of stalk sequence does not affect the direction of dynein movement. (A) Model of native dynein stalk (at left), showing the pair of conserved prolines (yellow spacefill) marking the MTBD and the conserved Trp-Gly pair that mark the top of the stalk coiled coil. Removal of seven heptads from the middle of the dynein stalk (−7) or insertion of seven heptads from the dynein stalk of Drosophila cytoplasmic dynein (+7) rotates the position of the Trp-Gly pair by 180° with respect to the microtubule-binding domain. (B) Cartoon of a model for how conformational changes in the AAA+ ring lead to minus end–directed motion [adapted from (8)] via rotation of the linker domain (yellow) toward the minus end. (C) Cartoon showing how the model in (B) predicts that rotation of the AAA+ ring by 180° due to a change in the length of the stalk should produce a plus end–directed motor. (D) Single-molecule fluorescence assay for the directionality of dimeric S. cerevisiae dynein constructs [based on GST-Dyn1_331kD (18)] with different lengths of stalk. Kymographs of tetra-methyl rhodamine–labeled dynein constructs from a single axoneme in the assay (green) show that all constructs move unidirectionally toward the minus end of the microtubule. The orientation of the axoneme was determined using fluorescent Cy5-labeled kinesin (see movies S2 to S4). Single-molecule velocities (means ± SD) and ATPase measurements (means ± SD) were determined as described in the supporting online material.

Fig. 4

Model for directional movement of dynein. (A) After release from the microtubule, the dynein is in a preconformational change (power stroke) state, with the linker domain (yellow) docked on the AAA+ domain (light blue circle) somewhat removed from the base of the stalk (gray). The rest of the dynein tail domain is represented as a loose spring attached to a cargo (red). The dynein MTBD (dark blue) is diffusing to a new site on the microtubule. (B) The MTBD preferentially enters a tightly bound state after binding toward the minus end, with the stalk at an angle. (C) An ATP-driven conformational change in the linker domain produces motion whose main vector directional component is parallel to the direction of the stalk (red arrow). The angle of the stalk thus converts this tension generated by the AAA+ domain into a displacement toward the minus end of the microtubule (as shown by the vector diagram), regardless of the orientation of the AAA+ ring.