|How Dynein Binds to Microtubules|
|Wednesday, 29 April 2009 00:00|
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.
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.
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.
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.
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).
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