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。微管相关的运动蛋白对于包括细胞迁移、有丝分裂、神经元分化和货物运输在内的多种生理功能是重要的，已经涉及许多疾病，从运动神经元和退行性疾病到肿瘤和病毒感染。微管结合CAP-Gly结构域在从人类到酵母的生物体中是保守的，在许多蛋白质中起核心作用，并且它们的突变导致各种疾病。动力蛋白p150 glued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相关。CAP-Gly/微管组装体的原子级结构和动力学尚不清楚，因为它们固有的不溶性和缺乏长程有序性。缺乏这种洞察力阻碍了进一步的研究，并阻碍了针对与细胞骨架功能障碍相关的疾病的有效疗法的设计。 我们的长期目标是了解在健康和疾病状态下，微管相关蛋白沿沿着进行货物运输调节的结构和动力学基础。本申请的目的是确定三维结构和动力学的CAP-Gly结构域的dynactin和它的大分子组件与微管和EB 1蛋白。我们将采用多维高分辨率魔角旋转固态核磁共振方法并结合生物物理和生化技术。在为实现本申请的目的而设计的具体目标中，我们将：1）确定单独的CAP-Gly和组装在微管上的CAP-Gly的结构，并以原子分辨率鉴定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）表征与神经病理学相关的CAP-Gly突变体的动力学; 4）从生物化学和结构上表征CAP-Gly/EB 1/微管相互作用的调节。拟议的工作对人类健康具有重要意义，因为它将揭示CAP-Gly的结构：微管复合物不适合通过X射线晶体学或溶液NMR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装体与微管复合物的结构表征成为可能。 公共卫生相关性：微管代表细胞中三种基本类型的细胞骨架之一，并且与其相关蛋白一起在广泛的生理功能中发挥重要作用，包括细胞迁移、有丝分裂、极化和分化以及囊泡和细胞器运输。微管相关蛋白与许多疾病有关，从运动神经元和退行性疾病到肿瘤和病毒感染。由于其固有的不溶性和缺乏长程有序性，微管组装及其相关蛋白质的原子分辨率结构和动力学尚不清楚。缺乏这种洞察力阻碍了进一步的研究，并阻碍了针对与细胞骨架功能障碍相关的疾病的有效疗法的设计。本申请中提出的研究将通过提供与微管结合的dynactin的p150 Glued亚基的微管相关CAP-Gly结构域的原子分辨率结构和动力学来填补这一知识空白。国家的最先进的固态核磁共振光谱将被引入作为一种新的技术来探测固有的不溶性和非结晶组装的微管与其相关的蛋白质。

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.