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(申请人提供)：微管代表细胞中三种基本的细胞骨架类型之一。微管相关运动蛋白具有多种重要的生理功能，包括细胞迁移、有丝分裂、神经元分化和货物运输，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。微管结合的CAP-Gly结构域在从人类到酵母的生物体中都是保守的，在许多蛋白质中发挥核心作用，它们的突变会导致各种疾病。动力蛋白p150粘合亚基的帽-甘氨酸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CAP-Gly/微管组装体本身的不溶性和缺乏长程有序性，其原子级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。我们的长期目标是了解在健康和疾病状态下，微管相关蛋白对沿微管的货物运输调节的结构和动力学基础。本应用的目的是确定dynactin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。我们将使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。为了实现这一应用的目标，我们将：1)确定单独的CAP-Gly和组装在微管上的CAP-Gly的结构，并在原子分辨率下确定CAP-Gly/微管界面；2)表征CAP-Gly/微管相互作用的能学和动力学；3)表征与神经病理相关的CAP-Gly突变体的动力学；4)从生化和结构上表征CAP-Gly/EB1/微管相互作用的调节。这项拟议的工作对人类健康具有重要影响，因为它将揭示CAP-Gly的结构：微管复合体不能通过X射线结晶学或溶液核磁共振光谱进行结构表征，并将能够对微管复合体中由微管相关蛋白组成的大分子组件进行结构表征。 与公共卫生相关：微管是细胞中三种基本的细胞骨架之一，与其相关的蛋白质一起，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，以及囊泡和细胞器的运输。微管相关蛋白与多种疾病有关，从运动神经元和退行性疾病到肿瘤和病毒感染。微管及其相关蛋白质的原子分辨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。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动力蛋白p150Glued亚单位的微管相关CAP-Gly结构域的原子分辨结构和动力学来填补这一知识空白。最先进的固态核磁共振波谱将作为一种新技术被引入，以探测微管及其相关蛋白质的本质上不可溶和非晶态的组装。