Conformation and recognition in microtubule dynamics

描述（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，是抗癌化疗药物如紫杉醇和长春花生物碱的直接靶点。MT的动态特性是其功能的核心，它们源于单个微管蛋白亚基的生化特性以及它们如何在MT晶格内相互作用。MT动力学是由一系列调控因子调控的，这些因子通常选择性地识别αß-微管蛋白的不同构象。αß-微管蛋白的两种不同构象已经在原子细节上被确定：一种与MT晶格兼容的“直”构象和一种与MT晶格不兼容的“弯”构象。通过对αß-微管蛋白组装的低分辨率研究揭示了αß-微管蛋白的不同构象，这些αß-微管蛋白组装模仿了在MT端观察到的独特几何形状。这些构象是否代表αß-微管蛋白的溶液构象？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αß-微管蛋白的默认构象来控制MT动力学？尽管进行了深入的研究，像这样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αß-微管蛋白本身或与MT相关蛋白（MAPs）复合物的原子结构极其困难。初步数据表明，现在有可能制备聚合阻断酵母αß-微管蛋白突变体，并利用它们确定αß-微管蛋白及其与调节蛋白复合物的新原子结构。这种独特的方法将允许新的实验来了解微管动力学的结构起源以及细胞因子如何调节它。目的1将确定酵母αß-微管蛋白和微管蛋白结合TOG结构域之间的复合物的结构，这些复合物来自微管动力学的基本调节因子，即含有多TOG的蛋白Stu2p。这将提供αß-微管蛋白与调节蛋白结合的第二种结构，并将为理解单个TOG结构域如何识别αß-微管蛋白提供结构框架。Aim 2将结合结构和生化方法来发现多个TOG结构域如何同时与αß-微管蛋白结合。这些实验将有助于更好地理解TOG结构域之间的协同作用如何有助于微管末端识别和促进Stu2p的伸长活性。Aim 3将通过确定与GTP或GDP结合的αß-微管蛋白的结构，并通过获得具有改变“曲率”的突变αß-微管蛋白，来回答有关未聚合αß-微管蛋白的构象及其如何依赖于核苷酸状态的问题。通过实现以前不可能的测量，并通过紧密结合结构和功能观察，这项工作的成功完成将代表对微管行为的结构决定因素的理解取得重大进展。

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 αß-tubulin. Two distinct conformations of αß-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 αß-tubulin were revealed by lower resolution studies of αß-tubulin assemblies that mimic the unique geometries observed at MT ends. Do any of these conformations represent the solution conformation of αß-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 αß-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 αß-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 αß-tubulin, and to use them to determine new atomic structures of αß-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 αß-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 αß-tubulin bound to a regulatory protein, and will provide a structural framework for understanding how individual TOG domains recognize αß-tubulin. Aim 2 will combine structural and biochemical approaches to discover how multiple TOG domains can bind to αß-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 αß-tubulin and how it depends on nucleotide state by determining structures of αß-tubulin bound to GTP or to GDP, and by obtaining mutant αß-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.