Conformation and recognition in microtubule dynamics

描述(申请人提供)：微管(MT)细胞骨架是真核细胞所必需的：微管是染色体分离和细胞内组织所必需的动态聚合物，是紫杉醇和长春花碱等抗癌化疗药物的直接靶标。MTS的动力学性质是其功能的核心，它们源于单个微管蛋白亚基的生化性质以及它们在MT晶格中的相互作用方式。微管动力学受一系列调节因子的调节，这些调节因子通常选择性地识别不同构象的微管蛋白。在原子细节上已经确定了两种不同的微管蛋白构象：一种是与MT晶格相容的“直”构象，另一种是与MT晶格不相容的“弯曲”构象。通过对模仿MT末端观察到的独特几何形状的β-微管蛋白组件的较低分辨率研究，发现了不同构象的？-微管蛋白。这些构象中有代表微管蛋白溶液构象的吗？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微管蛋白的默认构象来控制MT的动态？尽管进行了密集的研究，但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力是有限的：聚合的趋势使得获得β-微管蛋白本身或与MT相关蛋白(MAP)的复合体的原子结构极其困难。初步数据表明，现在有可能制备酵母β-微管蛋白的聚合阻断突变体，并用它们来确定β-微管蛋白及其与调节蛋白的络合物的新原子结构。这种独特的方法将使新的实验能够理解微管动力学的结构起源以及细胞因素是如何调节它的。目的1确定酵母β-微管蛋白与微管蛋白结合的TOG结构域之间的复合体的结构，该TOG结构域是微管动力学的重要调节因子，含有多TOG蛋白Stu2p。这将提供与调节蛋白结合的β-微管蛋白的第二个结构，并将为理解单个TOG结构域如何识别？-微管蛋白提供一个结构框架。目标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？-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Stu2p的微管末端识别和延长促进活性。目的3将通过测定与GTP或GDP结合的β-微管蛋白的结构，并获得具有改变“曲率”的突变的？-微管蛋白，来回答有关未聚合的β-微管蛋白的构象以及它如何依赖于核苷酸状态的问题。通过使以前不可能进行的测量成为可能，并通过将结构和功能观察紧密结合起来，这项工作的成功完成将代表着在理解微管行为的结构决定因素方面的重大进步。)

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. )