Structure and Dynamics of CAP-GLY: Microtubule Assemblies by Solid-State NMR

描述（由申请人提供）：微管代表细胞中三种基本细胞骨架类型之一。微管相关运动蛋白是多种生理功能的重要组成部分，包括细胞迁移、有丝分裂、神经元分化和货物运输，微管相关运动蛋白与许多疾病有关，包括运动神经元和退行性疾病、肿瘤和病毒感染。微管结合的CAP-Gly结构域在从人类到酵母的生物中都是保守的，在许多蛋白质中起着核心作用，它们的突变导致各种疾病。动力蛋白p150glue亚基的CAP-Gly结构域与微管相互作用，其突变与几种运动神经元疾病有关。由于其固有的不溶性和缺乏长程有序性，CAP-Gly/微管组件的原子水平结构和动力学尚不清楚。缺乏这样的认识阻碍了进一步的研究，也阻碍了设计针对与细胞骨架功能障碍相关疾病的有效疗法。我们的长期目标是了解微管相关蛋白在健康和疾病状态下沿微管运输货物的结构和动态基础。本应用程序的目的是确定动力学蛋白的CAP-Gly结构域及其与微管和EB1蛋白的大分子组装的三维结构和动力学。我们将采用多维高分辨率魔角旋转固态核磁共振方法结合生物物理和生化技术。为了实现本应用的具体目标，我们将：1)确定单独的CAP-Gly和组装在微管上的CAP-Gly的结构，并在原子分辨率上识别CAP-Gly/微管界面；2)表征CAP-Gly/微管相互作用的能量学和动力学；3)表征与神经病理相关的CAP-Gly突变体的动态；4)表征CAP- Gly/EB1/微管相互作用的生物化学和结构调控。拟议的工作对人类健康具有重要意义，因为它将阐明CAP-Gly：微管复合物的结构，这种结构无法通过x射线晶体学或溶液核磁共振光谱进行结构表征，并将使由微管相关蛋白组成的大分子组合物与微管复合物的结构表征成为可能。

DESCRIPTION (provided by applicant): Microtubules represent one of the three essential cytoskeleton types in cells. Important for a variety of physiological functions, encompassing cell migration, mitosis, neuronal differentiation and transport of cargo, microtubule-associated motor proteins have been implicated in numerous diseases, ranging from motor neuron and degenerative disorders, to neoplasia and viral infections. Microtubule-binding CAP-Gly domains are conserved in organisms from human to yeast, play central roles in many proteins, and their mutations lead to various disorders. CAP-Gly domain of the p150glued subunit of dynactin interacts with microtubules, and its mutations are associated with several motor neuron disorders. The atomic-level structure and dynamics of CAP-Gly/microtubule assemblies are not known because of their inherent insolubility and lack of long-range order. Lack of such insight hampers further research and impedes design of effective therapies against diseases associated with cytoskeleton dysfunction. Our long-term goal is to understand the structural and dynamic basis of cargo transport regulation along microtubules by microtubule-associated proteins, in healthy and disease states. The objectives of this application are to determine three-dimensional structures and dynamics of CAP-Gly domain of dynactin and of its macromolecular assemblies with the microtubules and with EB1 protein. We will employ multidimensional high-resolution magic angle spinning solid-state NMR methods in conjunction with biophysical and biochemical techniques. In the specific aims designed to accomplish the objectives of this application, we will: 1) determine the structure of CAP-Gly alone and CAP-Gly assembled on the microtubule, and identify the CAP-Gly/microtubule interface at atomic resolution; 2) characterize the energetics and dynamics of the CAP-Gly/microtubule interaction; 3) characterize the dynamics of CAP-Gly mutants related to neurological pathologies; 4) characterize biochemically and structurally the regulation of the CAP- Gly/EB1/microtubule interaction. The proposed work has important implications for human health as it will shed light on the structure of CAP-Gly:microtubule complexes that are not amenable to structural characterization by X-ray crystallography or solution NMR spectroscopy, and will enable structural characterization of macromolecular assemblies consisting of microtubule-associated proteins in complexes with microtubules. PUBLIC HEALTH RELEVANCE: Microtubules represent one of the three essential types of cytoskeleton in cells and, together with their associated proteins, play important roles in a broad range of physiological functions, encompassing cell migration, mitosis, polarization and differentiation, and vesicle and organelle transport. Microtubule-associated proteins have been implicated in numerous diseases ranging from motor neuron and degenerative disorders, to neoplasia and viral infections. Atomic-resolution structures and dynamics of microtubule assemblies with their associated proteins are not known due to their intrinsic insolubility and lack of long range order. Lack of such insight hampers further research and impedes design of effective therapies against diseases associated with cytoskeleton dysfunction. The research proposed in this application will fill this knowledge gap by providing the atomic-resolution structure and dynamics of the microtubule-associated CAP-Gly domain of the p150Glued subunit of dynactin bound to the microtubules. State-of-the-art solid-state NMR spectroscopy will be introduced as a novel technique to probe the intrinsically insoluble and non-crystalline assemblies of microtubules with their associated proteins.