Conformation and recognition in microtubule dynamics

微管动力学中的构象和识别

• 批准号: 8290307
• 负责人: Luke W Rice
• 金额: $ 30.19万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8290307
• 关键词: Achievement; Affect; Animals; Behavior; Binding; Biochemical; Biological Process; Brain; Cells; Chromosome Segregation; Complex; Conflict (Psychology); Cytoskeleton; DNA Sequence Rearrangement; Data; Disputes; Drug usage; Eukaryotic Cell; GTP Binding; Genetic Materials; Growth; Guanosine Triphosphate; In Vitro; Individual; Kinetics; Lead; Link; Malignant Neoplasms; Measurement; Measures; Mediating; Microtubule-Associated Proteins; Microtubules; Molecular; Molecular Conformation; Mutation; Nucleotides; Paclitaxel; Pharmaceutical Preparations; Plus End of the Microtubule; Polymers; Positioning Attribute; Property; Proteins; Reagent; Regulation; Resolution; Roentgen Rays; Saccharomyces cerevisiae; Series; Site; Solutions; Staging; Structure; System; Testing; Tubulin; Tubulin Interaction; Vinca Alkaloids; Work; X-Ray Crystallography; Yeasts; cell growth regulation; design; expectation; genetic regulatory protein; in vivo; innovation; insight; milligram; mutant; novel strategies; overexpression; polymerization; prevent; protein complex; research study; segregation

DESCRIPTION (provided by applicant): The microtubule (MT) cytoskeleton is essential to eukaryotic cells: microtubules are dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the biochemical properties of individual tubulin subunits and how they interact within the MT lattice. MT dynamics are modulated by a host of regulatory factors that often selectively recognize different conformations of ??-tubulin. Two distinct conformations of ?? -tubulin have been determined in atomic detail: a 'straight' one compatible with the MT lattice, and a 'curved' one that is not. Still different conformations of ?? -tubulin were revealed by lower resolution studies of ?? -tubulin assemblies that mimic the unique geometries observed at MT ends. Do any of these conformations represent the solution conformation of ?? -tubulin? How do conformation and conformational change contribute to the dynamic properties of the MT? Do regulatory proteins control MT dynamics by altering the default conformation of ?? - tubulin? Despite intense study, fundamental questions like these remain unresolved. Structural insight into these and other questions is limited: the tendency to polymerize makes it extremely difficult to obtain atomic structures of ?? -tubulin by itself or in complex with MT associated proteins (MAPs). Preliminary data demonstrate that it is now possible to prepare polymerization-blocked mutants of yeast ?? -tubulin, and to use them to determine new atomic structures of ?? -tubulin and its complexes with regulatory proteins. This unique approach will allow new experiments to understand the structural origins of microtubule dynamics and how cellular factors regulate it. Aim 1 will determine the structure of a complex between yeast ?? -tubulin and a tubulin-binding TOG domain from an essential regulator of microtubule dynamics, the multi-TOG containing protein Stu2p. This will provide the second-ever structure of ?? -tubulin bound to a regulatory protein, and will provide a structural framework for understanding how individual TOG domains recognize ?? -tubulin. Aim 2 will combine structural and biochemical approaches to discover how multiple TOG domains can bind to ?? -tubulin simultaneously. These experiments will lead to a better understanding of how cooperativity between TOG domains contributes to the microtubule end recognition and elongation promoting activities of Stu2p. Aim 3 will answer questions about the conformation of un polymerized ?? -tubulin and how it depends on nucleotide state by determining structures of ?? -tubulin bound to GTP or to GDP, and by obtaining mutant ?? -tubulin with altered 'curvature'. By enabling previously impossible measurements and by closely integrating structural and functional observations, successful completion of this work will represent a major advance in the understanding of the structural determinants of microtubule behavior. )

描述（由申请人提供）：微管（MT）细胞骨架对于真核细胞至关重要：微管是染色体分离和细胞内组织所需的动态聚合物，并且是紫杉醇和长春花生物碱等抗癌化疗药物的直接靶点。 MT 的动态特性是其功能的核心，它们源自各个微管蛋白亚基的生化特性以及它们在 MT 晶格内的相互作用方式。 MT 动力学受到许多调节因子的调节，这些调节因子通常选择性地识别 β-微管蛋白的不同构象。 ?? 的两种不同构象-微管蛋白已在原子细节上得到确定：一种与 MT 晶格兼容的“直”微管蛋白，一种与 MT 晶格不相容的“弯曲”微管蛋白。仍然有不同的构象？ -微管蛋白是通过较低分辨率的研究揭示的？ -模仿在 MT 末端观察到的独特几何形状的微管蛋白组件。这些构象中的任何一个都代表 ?? 的溶液构象吗？ -微管蛋白？构象和构象变化如何影响 MT 的动态特性？调节蛋白是否通过改变默认构象来控制 MT 动力学？ - 微管蛋白？尽管进行了大量的研究，诸如此类的基本问题仍然没有得到解决。对这些和其他问题的结构洞察是有限的：聚合的趋势使得获得 ?? 的原子结构变得极其困难。 -微管蛋白本身或与 MT 相关蛋白 (MAP) 复合。初步数据表明，现在可以制备聚合阻断的酵母突变体？ -微管蛋白，并使用它们来确定 ?? 的新原子结构-微管蛋白及其与调节蛋白的复合物。这种独特的方法将使新的实验能够了解微管动力学的结构起源以及细胞因素如何调节它。目标 1 将确定酵母 ?? 之间复合物的结构-微管蛋白和微管蛋白结合 TOG 结构域，来自微管动力学的重要调节剂，即含有多 TOG 的蛋白质 Stu2p。这将提供 ?? 的第二个结构。 -微管蛋白与调节蛋白结合，并将提供一个结构框架来理解各个 TOG 结构域如何识别？ -微管蛋白。目标 2 将结合结构和生化方法来发现多个 TOG 结构域如何结合？ -同时微管蛋白。这些实验将有助于更好地理解 TOG 结构域之间的协同性如何有助于 Stu2p 的微管末端识别和伸长促进活性。目标 3 将回答有关未聚合 ?? 构象的问题-微管蛋白以及它如何通过确定 ?? 的结构来依赖于核苷酸状态-微管蛋白与GTP或GDP结合，并通过获得突变体?? -“曲率”改变的微管蛋白。通过实现以前不可能的测量以及紧密结合结构和功能观察，这项工作的成功完成将代表着对微管行为的结构决定因素的理解的重大进展。 ）