Conformation and recognition in microtubule dynamics

描述（由申请人提供）：微管（MT）细胞骨架对真核细胞至关重要：微管是染色体分离和细胞内组织所需的动态聚合物，并且是抗癌化疗药物（如紫杉醇和紫堇生物碱）的直接靶点。MT的动力学性质是其功能的核心，并且它们来源于单个微管蛋白亚基的生化性质以及它们如何在MT晶格内相互作用。MT动力学是由一系列调节因子调节的，这些调节因子通常选择性地识别不同构象的？？微管蛋白两种不同的构象？- 微管蛋白已确定在原子细节：一个'直'的兼容MT晶格，和一个'弯曲'的，这不是。还是不同的构象？- 微管蛋白的低分辨率研究显示？- 微管蛋白组件，其模拟在MT末端观察到的独特几何形状。这些构象中是否有代表？的溶液构象？- 微管蛋白？构象和构象变化如何影响MT的动力学性质？调节蛋白是否通过改变？的默认构象来控制MT动力学？- 微管蛋白？尽管进行了大量的研究，但这些基本问题仍然没有得到解决。对这些问题和其他问题的结构洞察是有限的：由于倾向于重复，因此很难获得？？- 微管蛋白本身或与MT相关蛋白（MAP）复合。初步数据表明，它现在是可能的，以制备聚合阻断突变体的酵母？？- 微管蛋白，并使用它们来确定新的原子结构？- 微管蛋白及其与调节蛋白的复合物。这种独特的方法将允许新的实验，以了解微管动力学的结构起源和细胞因子如何调节它。- 微管蛋白和微管蛋白结合TOG结构域，所述TOG结构域来自微管动力学的必需调节剂，含有多TOG的蛋白Stu 2 p。这将提供有史以来第二个结构？？- 微管蛋白结合的调节蛋白，并将提供一个结构框架，了解如何个别TOG域识别？- 微管蛋白。目标2将结合联合收割机结构和生物化学的方法来发现多个TOG结构域如何结合？？- 微管蛋白。这些实验将导致更好地理解TOG结构域之间的协同性如何有助于微管末端识别和Stu 2 p的延伸促进活性。目标3将回答有关未聚合的？？- 微管蛋白和它如何取决于核苷酸状态，通过确定结构？- 微管蛋白结合GTP或GDP，并通过获得突变？- 具有改变的“曲率”的微管蛋白。通过使以前不可能的测量和紧密结合的结构和功能的观察，这项工作的成功完成将代表一个重大的进步，在微管行为的结构决定因素的理解。)

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. )