Structure and Mechanics of Dynein Motors

Abstract

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.

Keywords: axoneme; cilia; dynein; intracellular transport; intraflagellar transport; microtubules.

Figure 1

The assembly and activation of the dynein-1 transport machinery. (a) The φ-particle conformation of the dynein-1 complex (PDB accession code 5NVU) (186). Dynein-1 is inhibited by the self-dimerization of the motor domains at multiple contact sites. (b) The structure of the dynactin complex (PDB accession code 5ADX) (186). (c) Dynein-1 forms a ternary complex with dynactin and a coiled-coil cargo adaptor. The dynein-1 tail binds to the Arp1 filament of dynactin. Due to the translational symmetry of the Arp1 filament, the dynein-1 HCs form a parallel orientation and walk processively along MTs. Dynactin recruits a second dynein-1 motor, which results in a faster and stronger motor complex. Insets represent a 180°-rotated view of ternary interactions among the dynein-1 tail, dynactin, and the N-terminal fragment of the BicD2 adaptor (PDB accession code 5AFU) (173). Abbreviations: HC, heavy chain; IC, intermediate chain; LC, light chain; LIC, light-intermediate chain; MT, microtubule; PDB, Protein Data Bank.

Figure 2

Model for plus-end recruitment of dynein-1. (a) Dynactin is recruited to the plus end by tip tracking proteins EB1 and CLIP-170. Lis1 binds to the dynein-1 motor domain and stabilizes the open conformation of dynein-1 in the cytoplasm. (b) Together with a cargo adaptor protein, dynactin recruits Lis1-bound dynein-1 in the open conformation to form the active complex. Following complex assembly, Lis1 dissociates from dynein as DDX moves processively toward the minus end of the MT. Abbreviations: DDX, dynein-1-dynactin-cargo adaptor; MT, microtubule.

Figure 3

The mechanochemical cycle of the dynein-1 motor domain. (State 0) Schematic representation of the dynein-1 motor domain. The AAA ring (subunits are numbered 1–6) is attached to the MTBD through a coiled-coil stalk. The linker resides at the surface of the ring and connects to the tail (not shown). The prerequisite of the mechanochemical cycle is ATP binding and hydrolysis at the AAA3 site. AAA3 remains in a posthydrolysis (i.e., ADP-bound) state to enable the nucleotide state of AAA1 to control the linker conformation and stalk registry. (State 1) In the apo state of AAA1, dynein-1 is bound to the MT, the coiled-coil stalk is in the α registry, and the linker is in the straight conformation. (State 2) Upon ATP binding at AAA1, AAA5–6 undergo rigid body motion (dashed arrow), which triggers the buttress to slide stalk coiled-coils relative to each other (solid arrow). (State 3) The stalk shifts to the β registry, and the motor releases from the MT. The linker is allowed to move freely across the surface of the ring. (State 4) Upon ATP hydrolysis, the linker converts to the bent conformation, and this priming stroke moves the MTBD toward the minus end. (State 5) The MTBD undergoes a diffusional search and rebinds to the MT lattice. MT binding triggers shifting of the stalk coiled-coils (solid arrow) and rigid body motion in the AAA ring (dashed arrows). (State 6) The stalk adopts the α registry. The inorganic phosphate is released from AAA1. The linker moves the straight conformation through the force-generating powerstroke. Following ADP release, the motor returns to the initial apo state (State 1). Abbreviations: MT, microtubule; MTBD, MT-binding domain.

Figure 4

Stepping, directionality, and force generation of dynein-1. (a) (Top right) The stepping of a QD-labeled DDX motor was tracked on surface-immobilized MTs. (Middle) Representative stepping traces (black dots) are fit to a step-finding algorithm (horizontal lines). (Bottom) The histogram reveals that dynein-1 takes steps that are highly variable in size and direction. Panel adapted from Reference . (b) (Top) Cryo-EM 2D class averages of WT and engineered yeast dynein-1 monomers. Arrows point to the N terminus of the linker. (Bottom) In engineered dynein-1, the angle that the stalk makes relative to the MT is reflected by shifting the positions of proline residues in both coiled-coils, and the AAA ring is flipped around the stalk axis by a seven-heptad insertion to the stalk coiled-coils (highlighted in yellow). These modifications reversed the direction of the linker swing and resulted in the plus end–directed motility (blue arrow). Panel adapted from Reference . (c) (Left) External force induces the release of the motor from the MT. The force alters the stalk registry such that the stalk coiled-coils are trapped in the strongly bound (α) registry under hindering forces, while they switch to the lower-affinity (γ) registry under assisting forces, resulting in faster release when dynein is pulled toward the minus end. (Right) The force-induced release rate of a dynein-1 monomer in the apo condition exhibits strong asymmetry that favors faster release under assisting force. Panel adapted from Reference . (d) (Left) DDX is attached to a bead held in an optical trap. (Right) An example trajectory shows that the motor pulls the bead against the trap. The resistive force increases until the motility stalls and the motor releases from the MT (red arrowhead). Panel adapted from Reference . Abbreviations: cryo-EM, cryogenic electron microscopy; DDX, dynein-1-dynactin-cargo adaptor; MT, microtubule; QD, quantum dot; WT, wild type.

Figure 5

Activation and recycling of dynein-2 in IFT. (a) (Top) The cross-section of an axoneme shows nine MT doublets surrounding a central pair of MTs. (Bottom) Organization of IFT trains on a single MT doublet (76). Anterograde trains are comprised of IFT-A and IFT-B complexes and transported by kinesin-II along B-tubules toward the ciliary tip. Dynein-2 is bound to IFT-B and remains in the auto-inhibited φ conformation. Trains offload their cargo and disassemble at the tip. Retrograde trains, formed by available IFT-B complexes at the tip, recruit and activate dynein-2 to move back to the ciliary base. In Chlamydomonas IFT, kinesin-2 detaches from IFT at the tip and diffuses inside the cilium. (b) The φ conformation of dynein-2. One of the HCs (DHC-A) has a straight tail conformation, while the tail of the other HC (DHC2-B) has a switch-back conformation that contacts with IFT-B complexes at multiple locations (not shown). The HCs are dimerized by the heterodimer of WDR34 and WDR60 ICs, which are connected by RB and three dimers of LC8 (PDB accession code 6SC2) (168). The schematic representation of the coiled-coil stalk and MTBD were added from PDB accession code 3VKH (86). Abbreviations: HC, heavy chain; IC, intermediate chain; IFT, intraflagellar transport; MT, microtubule; MTBD, MT-binding domain; PDB, Protein Data Bank.

Figure 6

Structure and regulation of axonemal dyneins. (a) (Left) A schematic representation of a spermatozoon. (Right) Ciliary cross-section displaying the arrangement of dyneins in the inner and outer arm of the axoneme. (b) Organization of OAD (red) and IAD (cyan) motors along adjacent MT doublets. OAD heterotrimers are spaced 24 nm apart with their MTBDs extending toward the adjacent B-tubule. IAD dyad pairs are organized in repeating arrays of (fα–fβ), (a–b), (c–e), (g–d) every 96 nm. (c) Models for dynein regulation during ciliary beating. In the geometric clutch model, ciliary bending changes the distance between adjacent MTs, which prevents the motors from attaching to the MT. In the curvature control model, ciliary bending generates a stretched lattice on the convex side. The OAD MTBD senses the changes in MT curvature. In the sliding control model, nexin linkers generate a resisting force (green arrow) parallel to the axoneme, causing motors to release from the MT at high forces. For clarity, only the transitions from straight to convex bends are displayed. Abbreviations: IAD, inner arm dynein; MT, microtubule; MTBD, MT-binding domain; OAD, outer arm dynein.