Topological Phonons in Microtubules: The Link between Local Structure and Dynamics of Microtubules

Abstract

Microtubules and their associated proteins are central to a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. They have been extensively studied at multiple levels of resolution. However, there remain significant gaps in knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states and how these interactions and dynamics affect the cellular processes. Structures of many microtubule-associated proteins assembled on microtubules are not available at atomic resolution. We used magic angle spinning (MAS) NMR to solve a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin assembled on microtubules. We determined the intermolecular interface of CAP-Gly with polymerized microtubules by direct dipolar contacts between CAP-Gly and tubulins using double rotational echo double resonance (dRE-DOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with microtubules and other binding partners. This study is the first example of the atomic-resolution structure of a microtubule-associated protein on polymerized microtubules. We have also characterized the structure and dynamics of the extended CAP-Gly domain that contains the CAP-Gly domain, basic domain and serineproline-rich (SP-rich) domain of p150 glued subunit by solution NMR and MAS NMR. Our results indicate that the basic and SP-rich domains contain a second binding site for microtubules in addition to CAP-Gly domain. The secondary structure prediction is performed based on backbone chemical shifts from solution NMR and three short segments in the basic domain are predicted to contain helical and sheet structures. These segments are contained in the rigid regions as revealed by the conformational flexibility analysis and possibly encompass the intermolecular binding sites for microtubules.