Crystal structure of the dynein motor domain

Abstract

Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein's large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ~300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an unusual asymmetric arrangement of ATPase domains in the ring-shaped motor domain, the manner in which the mechanical element interacts with the ATPase ring, and an unexpected interaction between two coiled coils that create a base for the microtubule binding domain. The arrangement of these elements provides clues as to how adenosine triphosphate-driven conformational changes might be transmitted across the motor domain.

Fig. 1

The cytoplasmic dynein motor domain crystal structure. (A) Schematic illustrating the domains of the dimeric yeast cytoplasmic dynein heavy chain. For crystallization, the linker was fused to GST, and the MTBD and part of the stalk (amino acids 3039 to 3291) were removed and replaced with a short peptide (20). (B) The complete GST-cytoplasmic dynein dimer; GST is in green. (C) View of the motor domain from the linker face. The linker, stalk, and C terminus are color-coded as in (A).

Fig. 2

The dynein AAA domains. (A) The six individual AAA domains are highlighted in colors. The linker spanning over the center of the ring is magenta. (B) Topology of the dynein large and small domains. (C) The electron density map [experimental (Fo) map contoured at 1 σ] and model build of the secondary structure elements (helices and central β sheet) of a dynein AAA large domain (AAA6L). (D) Experimental electron density map and model showing a unique helical insert (blue) in AAA4L. Insert sequences are shown in fig. S5. (E) Experimental electron density map and model of a dynein small domain (AAA1s). Omit maps of AAA1 and AAA2 small domains are shown in fig. S1. (F and G) Comparison of the large and small domains of AAA1 to 3. Galleries for all large and small domains can be found in figs. S3 and S4, respectively.

Fig. 3

Asymmetry of the dynein ring. (A) Side view of AAA ring showing the different planes occupied by the AAA1 to 3 large domains. (B) View of the linker face of the AAA ring (as in Fig. 2A), showing just the large domains. Note the large gaps between AAA1 and 2 and between AAA5 and 6. ATP is expected to bind between AAA1 and 2. (C) Comparison of the positions of the small domains relative to the large domain for AAA1, 3, and 6 (large domains aligned to AAA1); small domains can adopt a variety of different orientations due to a flexible linker joining β5 (large) to H5 (small). (D) Packing of the small domains against the neighboring AAA large domain. (Inset) AAA1s against AAA2L, AAA3s against AAA4L, and AAA6s against AAA1L. AAA large domains were aligned to AAA1. The orientations of the small domains to the neighboring large domain are similar to one another. (E) Comparison of the positions of adjacent large domains in dynein and ClpX (PDB code 3HWS). In the nucleotide-bound ClpX monomers in the hexamer (monomer chain A shown here) and AAA3 of dynein (aligned with ClpX), the adjacent large domain is closely apposed in a closed conformation. (F) In the nucleotide-free monomers of the same ClpX hexamer (monomer chain C) and AAA1, the adjacent large domain is more widely separated in an open conformation, due to the rotation of the intervening small domain. Comparison of the positions of adjacent large domains around the dynein AAA ring is found in fig. S7.

Fig. 4

The linker domain and its interaction with the AAA ring. The linker and its four helical subdomains (subdomains 1 and 4 are N- and C-terminal, respectively) are indicated, and the contact sites (<8 Å) with the AAA domains are shown in color on the space-filling model.

Fig. 5

The stalk and buttress coiled coils. (A) The motor domain, highlighting the stalk and buttress, viewed from the C-terminal face. The stalk from this crystal structure is highlighted in yellow, and the green extension is a continuation of the stalk modeled with an antiparallel coiled coil of the proper length. The MTBD and distal coiled coil is from a previously solved crystal structure (PDB code 3ERR). (B) The experimental electron density map (1 σ contour) and model showing the likely interaction of the distal part of the buttress with the stalk. (C) The small domains of AAA4 and AAA5 show that H7 and H8 extend into the stalk coiled coil, and H5 and H6 extend into the buttress coiled coil.

Fig. 6

Models for dynein conformational changes. (A) A schematic model showing how ATP binding to AAA1 might propagate a conformation change to the MTBD and linker (in white). (Left) The apo state with gaps between AAA1 and 2 and between AAA5 and 6, as based on our crystal structure (Fig. 3B). (Right) The proposed consequence of ATP binding to AAA1: closure of the AAA1-2 gap, which in turn pulls upon and moves AAA2, 3, and 4. The relative motions that might ensue between AAA4 and 5 cause the detachment of the linker from AAA5. The indicated position of the linker in the ATP state is based on EM studies of Roberts et al. (8), although it may be mobile and not have a defined docked state on the ring. AAA4 and 5 movement may create a shear between the stalk (yellow coiled coil) and buttress (orange coiled coil); if the stalk preferentially interacts with the buttress through one of its helices, this could shift the registry of the stalk helices (arrow) that propagates to the MTBD to change its affinity (18, 34). (B) (Left) The dynein crystal structure with the distal stalk and MTBD, modeled as in Fig. 5A. One of the MTBDs is docked onto the microtubule as determined previously (18). (Right) One possible model in which the second head (light orange) has its MTBD docked onto the microtubule. To allow this head and its MTBD to bind to the microtubule, its linker was undocked from AAA5 and rotated about subdomain 3, and a slight bend was applied to the stalk of the front (left) head.