How Dynein Binds to Microtubules

Cytoplasmic dynein is a protein complex responsible for the transport of a large variety of cargoes, from specific RNAs and proteins to whole organelles, in a directional fashion along microtubules that serve as cellular conveyor belts. Consistent with this central role, cytoplasmic dynein is associated with a number of disease-related processes, including the transport of viruses, neurodegeneration, and the mitotic checkpoint malfunctions that lead to cancer. A team of researchers from the University of California's San Francisco and Berkeley campuses has recently solved the structure of dynein's microtubule-binding domain (MTBD) and part of the stalk structure that connects MTBD to the rest of the dynein complex. This first look at any part of the dynein motor domain identifies how it binds to microtubules and gives some hints into the fascinating question of how communication passes along the stalk from the MTBD to the rest of the motor.

Lucky Break
Gives New Legs to Research

Motor proteins are excellent examples of "nanotechnology" in Nature. These fascinating protein molecules convert the chemical energy stored in adenosine triphosphate (ATP) molecules into mechanical work. Some motor proteins, such as myosin, are found in muscle fibers, and their functioning ultimately results in macroscopically observable motion, such as a person walking down a hall. Other motor proteins work on the cellular level, transporting various cargoes such as chromosomes during cell division or positioning organelles such as the Golgi complex. Dynein, along with kinesin (see previous ALS highlight, "Kinesin Action Crystallized in Two Key States"), are examples of such motor proteins whose "bipedal" motion mimics the way a person walks down a hall. These proteins typically have two "legs" that alternately bind to and release from a cellular path as they haul their cargoes to their destinations—kinesin moving in one direction and dynein in the other. In this work, Carter et al. focus on part of one "foot" of the dynein complex. After almost two years of work on crystallizing this construct, the researchers got a lucky break when a new technician spent extra minutes examining the crystals before freezing them in preparation for analysis, allowing them to dehydrate a bit and thereby greatly improving their ability to diffract.

Drawing of the dynein structure, showing the AAA+ ring of the motor domain attached to the dynein MTBD by an antiparallel coiled coil of alpha helices (the "stalk"). The boxed region indicates the structure solved in this study. Drawing source: R.D. Vale, Cell 112, 467 (2003).

The heart of cytoplasmic dynein is the motor domain that couples hydrolysis of ATP with conformational changes that drive motility and that remains the only major class of cytoskeletal motors for which crystal structures are not available. The motor domain is built out of a ring of six protein domains belonging to the AAA+ family and binds to microtubules by a specialized binding domain found at the end of a long, thin stalk that emerges from between two of the AAA+ domains.

In order to produce a stable construct for crystallization, the dynein MTBD and the top part of the stalk, which forms an antiparallel coiled coil of two alpha helices, were fused into the coiled coil of a known protein called seryl-tRNA synthetase. Molecular replacement using the seryl-tRNA synthetase part of the fusion protein revealed sufficient density of the dynein MTBD to allow its structure to be solved to a resolution of 2.3 angstroms at ALS Beamline 8.3.1.

Isolated dynein MTBD. The coiled-coil stalk alpha helices are shown in red and purple, with the conserved prolines that cause a kink shown as red spheres. The microtubule-interacting surface is made up of alpha helices H1, H3, and H6.

The observed structure confirms the coiled-coil nature of the dynein stalk and shows that it is kinked by the presence of two highly conserved prolines (amino acids). The rest of the MTBD consists of a novel fold of alpha helices that pack against the top of the stalk. The top face (including helices H1, H3, and H6) was identified as the site of interaction with microtubules, based on mutations that interfere with binding. Confirmation was provided by docking the crystal structure into an electron-density map of a dynein stalk bound to microtubules that was obtained by cryoelectron microscopy.

Left: Cryoelectron microscope image of dynein MTBDs (blue) bound to a microtubule (green). Right: MTBD and tubulin structures docked into an electron-density map (blue mesh) of a single microtubule protofilament decorated with dynein MTBDs.

In order for dynein to move cargoes, the conformational changes in the AAA+ ring that drive movement must be correctly coupled to its binding and unbinding from microtubules. This coupling requires communication between the MTBD and AAA+ ring. The researchers gained insight into this process by noticing that the affinity of the MTBD could be controlled by changing the fusion site in the seryl-tRNA synthetase fusion protein. This observation led them to suggest that communication was mediated by a small (four-amino-acid) relative sliding of the alpha helices in the stalk. A number of features of the structure are consistent with this mechanism. For example, the top of the helix that is thought to slide during communication makes relatively few contacts with the rest of the MTBD but connects directly to the main helix involved with contacting microtubules. In contrast, the other stalk helix makes extensive contacts with the rest of the MTBD, consistent with its role as a more rigid element against which the other stalk helix slides.

While the MTBD structure solved at the ALS is but a step on the road toward understanding dynein's function, it is already beginning to provide mechanistic insight.

Four of the authors of the paper at Beamline 8.3.1 (shortly after data collection). On the left are Andrew Carter and Joan Garbarino (the two joint first authors), behind Joan is Wes Shipley (the technician whose crystal freezing advanced the project) and on the far right is Ian Gibbons, a senior author on the paper and the first person to isolate (and name) dynein in 1965.

Research conducted by A.P. Carter, C. Cho, and R.D. Vale (Howard Hughes Medical Institute and University of California, San Francisco); J.E. Garbarino, W.E. Shipley, and I.R. Gibbons (University of California, Berkeley); and E.M. Wilson-Kubalek and R.A. Milligan (The Scripps Research Institute).

Research funding: Jane Coffin Childs Foundation, National Institutes of Health, Agouron Institute, Leukemia and Lymphoma Society, and Howard Hughes Medical Institute. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: A.P. Carter, J.E. Garbarino, E.M. Wilson-Kubalek, W.E. Shipley, C. Cho, R.A. Milligan, R.D. Vale, and I.R. Gibbons, "Structure and functional role of dynein's microtubule-binding domain," Science 322, 1691 (2009).

ALSNews Vol. 297, April 29, 2009

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