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

基本信息

批准号：
7895145
负责人：
Tatyana Polenova
金额：
$ 39.14万
依托单位：
UNIVERSITY OF DELAWARE
依托单位国家：
美国
项目类别：
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/7895145
关键词：
Abbreviations Address Affect Affinity Amyotrophic Lateral Sclerosis Area Arts 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 novel public health relevance research study solid state nuclear magnetic resonance structural biology three dimensional structure

项目摘要

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基本细胞骨架类型之一。微管相关运动蛋白对于多种生理功能（包括细胞迁移、有丝分裂、神经元分化和货物运输）很重要，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 CAP-Gly 结构域在从人类到酵母的生物体中是保守的，在许多蛋白质中发挥着核心作用，它们的突变会导致各种疾病。 dynactin p150glued 亚基的 CAP-Gly 结构域与微管相互作用，其突变与多种运动神经元疾病相关。由于 CAP-Gly/微管组件固有的不溶性和缺乏长程有序，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下微管相关蛋白沿微管的货物运输调节的结构和动态基础。本应用的目的是确定 dynactin 的 CAP-Gly 结构域及其与微管和 EB1 蛋白的大分子组装体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与生物物理和生化技术相结合。为了实现本申请的具体目标，我们将：1）确定单独的CAP-Gly和组装在微管上的CAP-Gly的结构，并以原子分辨率识别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）表征与神经病理学相关的CAP-Gly突变体的动态； 4) 从生化和结构上表征 CAP-Gly/EB1/微管相互作用的调节。这项工作对人类健康具有重要意义，因为它将揭示 CAP-Gly:微管复合物的结构，这些复合物不适合通过 X 射线晶体学或溶液核磁共振光谱进行结构表征，并且将能够对由以下物质组成的大分子组装体进行结构表征：与微管复合物中的微管相关蛋白。公共健康相关性：微管代表细胞中三种基本细胞骨架类型之一，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。由于微管组件固有的不溶性和缺乏长程有序，其原子分辨率结构和微管组件及其相关蛋白质的动力学尚不清楚。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。最先进的固态核磁共振波谱将作为一种新技术来探测微管及其相关蛋白质的本质不溶性和非晶体组件。