Microtubule-associated CAP-Gly domain of dynactin: structure, dynamics, conformational plasticity, and interactions with microtubules and microtubule plus-end tracking proteins by magic angle spinning NMR spectroscopy
Date
2014
Authors
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Publisher
University of Delaware
Abstract
Microtubules and their associated proteins play essential roles in a broad range of physiological functions, including cell migration, mitosis, polarization, differentiation, and vesicle and organelle transport. The malfunctions of microtubule-associated proteins are related to numerous diseases. Despite the importance of this system, the mechanisms of the interactions between microtubules and their associated proteins are still not fully understood, especially on the atomic-resolution level. The assemblies formed by microtubules and their associated proteins are large, insoluble and lack long-range order, which precludes their atomic-level structure and dynamic characterization by the two most prevalent structural biology techniques, X-ray crystallography and solution NMR spectroscopy. ☐ Magic angle spinning (MAS) NMR spectroscopy is an approach uniquely suited for gaining atomic-level information into the structure and dynamics of assemblies of microtubules with their associated proteins, because it does not require solubility or long-range order, and there are no limitations on the molecular weight of the system under investigation. The focus of this dissertation is gaining comprehensive structural and dynamics insights into the p150Glued CAP-Gly domain of mammalian dynactin, and understanding its interactions with microtubules and microtubule plus-end tracking proteins, by MAS NMR spectroscopy. ☐ Using MAS NMR spectroscopy, we have obtained high-quality three-dimensional structures of the CAP-Gly domain in the unbound state (Chapter 3). We have investigated the interactions of the CAP-Gly domain with a microtubule plus-end tracking protein EB1 (Chapter 4). We have solved the first atomic-resolution 3D structure of the CAP-Gly assembled on microtubules (Chapter 5). In addition to the structural studies of the CAP-Gly domain, we have performed internal dynamics studies of free CAP-Gly, CAP-Gly assembled on microtubules and CAP-Gly in complex with EB1 (Chapter 6). With these structural and dynamic investigations of free CAP-Gly and CAP-Gly in complex with microtubules or with its binding partner, we have discovered the unique conformational plasticity of the CAP-Gly domain, which we postulate to be essential for its ability to walk along microtubules and to interact with its various binding partners to regulate microtubule dynamics. ☐ The studies detailed in this dissertation serve as an important step towards our long-term goal, which is to decipher the regulation mechanisms of microtubule-based intracellular transport mechanisms at atomic resolution, using MAS NMR spectroscopy and other biophysical techniques.