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

CAP-GLY 的结构和动力学：通过固态 NMR 观察微管组件

• 批准号: 8050102
• 负责人: Tatyana Polenova
• 金额: $ 30.69万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050102
• 关键词: Abbreviations; Address; Affect; Affinity; Amyotrophic Lateral Sclerosis; Area; Avidity; Binding; Biochemical; Biophysics; COX7A2L Protein; Cells; Cellular biology; Chemicals; Chromosomes; Cities; Coiled-Coil Domain; Collaborations; Complex; Cytoskeleton; Data; Degenerative Disorder; Delaware; Disease; Dynein ATPase; Functional disorder; Goals; Health; Human; Investigation; Kinetochores; Knowledge; Lead; Light; Magic; Manuscripts; Measurement; Mediating; Methodology; Methods; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Missense Mutation; Mitosis; Mitotic Checkpoint; Modification; Motor; Motor Neurons; Mutation; NMR Spectroscopy; Neoplasms; Neuronal Differentiation; Organelles; Organism; Paclitaxel; Patients; Physiological; Play; Process; Property; Protein Binding; Protein Biochemistry; Proteins; Regulation; Relaxation; Research; Research Institute; Resolution; Role; Series; Serine; Signal Transduction; Signaling Molecule; Solutions; Spinobulbar Muscular Atrophy; Structure; Syndrome; Techniques; Testing; Time; Translations; Tubulin; Tumor Suppressor Proteins; Universities; Vesicle; Virus Diseases; Work; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; base; cell motility; cofactor; design; dynactin; dynein light chain; effective therapy; in vivo; insight; macromolecular assembly; molecular dynamics; mutant; neurological pathology; neuronal transport; novel; public health relevance; research study; solid state nuclear magnetic resonance; structural biology; three dimensional structure

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.

描述（由申请人提供）：微管代表细胞中三种必需的细胞骨架类型之一。对于各种生理功能，重要的是细胞迁移，有丝分裂，神经元分化和货物的运输，微管相关的运动蛋白与多种疾病有关，从运动神经元和退化性疾病到新自动疾病和病毒感染。微管结合的帽盖结构域在从人到酵母菌的生物中保守，在许多蛋白质中起着核心作用，它们的突变导致了各种疾病。 Dynactin的P150GLOUD亚基的帽盖域与微管相互作用，其突变与几种运动神经元疾病有关。瓶盖/微管组件的原子级结构和动力学因其固有的不溶性和缺乏远距离顺序而尚不清楚。缺乏这种洞察力的篮筐进一步研究，并阻碍了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健康和疾病状态中与微管相关蛋白沿微管沿微管沿着微管调节的结构和动态基础。该应用的目的是确定Dynactin及其大分子组件的三维结构和与微管和EB1蛋白的大分子组件的动力学。我们将采用多维高分辨率魔术角旋转固态NMR方法以及生物物理和生化技术。在旨在实现此应用程序目标的特定目的中，我们将：1）确定在微管上单独使用帽盖的结构，并在微管上组装，并在原子分辨率下识别帽帽/微管界面； 2）表征瓶盖/微管相互作用的能量和动力学； 3）表征与神经病理学有关的帽旋转突变体的动力学； 4）从生化和结构上表征盖胶/EB1/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，因为它将阐明帽胶的结构：微管复合物，这些复合物不适合通过X射线晶体学或溶液NMR光谱法进行结构表征，并将实现大分子组件的结构表征与微管的复合物中的微管相关蛋白。 公共卫生相关性：微管代表细胞中三种基本细胞骨架的一种类型之一，以及它们相关的蛋白质，在广泛的生理功能中起着重要作用，包括细胞迁移，有丝分裂，极化和分化以及囊泡和有机体的运输。微管相关的蛋白质与许多疾病有关，从运动神经元和退化性疾病到肿瘤和病毒感染。原子分辨率结构和及其相关蛋白的微管组件的动力学因其内在的不溶性和缺乏远距离顺序而尚不清楚。缺乏这种洞察力的篮筐进一步研究，并阻碍了与细胞骨架功能障碍相关的疾病的有效疗法的设计。本应用程序中提出的研究将填补这一知识差距，通过提供与微管的p150glued亚基的微管相关的帽盖域的原子分辨率结构和动力学。最先进的固态NMR光谱将作为一种新型技术引入，以探测微管与其相关蛋白质的本质上不溶性和非晶体组件。