CONFORMATION & RECOGNITION IN MICROTUBLE DYNAMICS

结构

• 批准号: 9355199
• 负责人: Luke W Rice
• 金额: $ 32.07万
• 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 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9355199
• 关键词: Address; Adopted; Affect; Behavior; Binding; Biochemical; Biochemistry; Cells; Chromosome Segregation; Coupling; Cryoelectron Microscopy; Crystallization; Defect; Engineering; Equilibrium; Frequencies; Genetic Materials; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; In Vitro; Kinetics; Laboratories; Link; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microtubule Polymerization; Microtubule Stabilization; Microtubules; Molecular; Molecular Conformation; Mutagenesis; Mutation; Negative Staining; Paclitaxel; Pharmaceutical Preparations; Polymerase; Polymers; Positioning Attribute; Property; Protein Engineering; Proteins; Reagent; Regulation; Role; Site; Structure; Surface; Testing; Time; Tubulin; Variant; Vinca Alkaloids; Work; Yeasts; alpha Tubulin; base; beta Tubulin; design; experimental study; genetic regulatory protein; insight; mutant; novel; polymerization; reconstitution; residence; response; segregation; self assembly; single molecule

Microtubules (MTs) are essential dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. It is increasingly appreciated that the polymerizing αβ-tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to promote elongation, shrinking, or catastrophe. However, integrating these structural and biochemical findings into a mechanistic understanding of MT dynamics and regulation remains a central challenge. In the prior project period, we pioneered a powerful approach based on structure-inspired site- directed αβ-tubulin mutants. Using these methods, we advanced the understanding of the regulatory MT polymerase Stu2p by discovering that two simple concepts – selective binding to a MT-incompatible conformation of αβ-tubulin and tethering-based concentration of reactants – could explain the catalytic action of the polymerase. We also advanced the understanding of MT dynamics, discovering by studying buried mutations in β-tubulin that a tunable allosteric response to GDP in the lattice dictates the frequency of MT catastrophe and the rate of post-catastrophe shrinking. Our laboratory is now uniquely positioned to answer fundamental questions about MT dynamics and regulation. In Aim 1 we will use a combination of protein engineering and in vitro reconstitution, including single molecule experiments, to answer most of the remaining questions about the mechanism of MT polymerases: the molecular origin of processivity, what determines the degree of polymerase saturation on the MT end, and how maximal polymerase activity depends on the number and type of TOG domains. This will result in a state-of-the-art understanding of a MT regulatory protein that integrates structure and biochemistry with bulk and single-molecule kinetic results In Aim 2 we capitalize on our finding that α:E255A, a surface mutation at the site of GTPase activity, causes a `straightening defect'. We will use mutagenesis, measurements of MT dynamics, negative stain and cryo electron microscopy, and other approaches to discover the mechanism of assembly-dependent αβ-tubilin straightening, identify allosteric coupling within the heterodimer, and show how they contribute to MT dynamics and the structure of αβ-tubulin assemblies. In Aim 3 we will use a stable of new reagents to determine αβ-tubulin structures without binding partners or bound to a TOG domain. This work will answer longstanding questions about conformation(s) of unpolymerized αβ-tubulin, will reveal whether allosteric mutations change it, and it will clarify what αβ-tubulin conformations can be recognized by TOG domains. Our approach is distinctive and promises to deliver previously unobtainable insight into fundamental mechanisms of MT dynamics and regulation.

微管是染色体分离和细胞内所必需的动态聚合物 组织，是抗癌化疗药物如紫杉醇和长春花碱的直接靶标。它是 人们越来越认识到，聚合的αβ-微管蛋白亚基采用不同的构象作为 GTP酶依赖聚合动力学，调节蛋白选择性地识别 这些构象促进伸长、收缩或灾难。然而，将这些结构性的 对MT动力学和调节的机械性理解的生化发现仍然是核心 挑战。在之前的项目期间，我们开创了一种基于结构灵感站点的强大方法- 定向αβ-微管蛋白突变体。利用这些方法，我们加深了对规范性MT的理解 聚合酶Stu2p发现两个简单的概念-选择性结合到MT-不相容 αβ的构象--微管蛋白和基于链结的反应物浓度--可以解释催化作用 聚合酶的。我们也加深了对MT动力学的理解，通过研究掩埋发现 β-微管蛋白的突变，晶格中对GDP可调节的变构反应决定了MT的频率 巨灾和巨灾后收缩的速度。我们的实验室现在处于独特的位置来回答 关于MT动态和监管的基本问题。在目标1中，我们将使用蛋白质的组合 工程和体外重建，包括单分子实验，以回答大多数 关于MT聚合酶机制的遗留问题：加工的分子起源，什么 确定MT端的聚合酶饱和程度，以及最大聚合酶活性 取决于TOG域的数量和类型。这将导致对MT的最先进的理解 将结构和生物化学与整体和单分子动力学相结合的调节蛋白导致 目标2我们利用我们的发现，α：E255A，GTP酶活性位点的表面突变，导致 ‘拉直缺陷’。我们将使用诱变、MT动态测量、阴性染色和冷冻 电子显微镜和其他方法发现依赖组装的αβ-tubilin的机制 矫直，确定杂二聚体中的变构偶联，并展示它们如何对MT做出贡献 αβ-微管蛋白组件的动力学和结构。在目标3中，我们将使用一系列新的试剂来 确定没有结合伙伴或与TOG域结合的αβ-微管蛋白结构。这项工作将回答 关于未聚合的αβ-微管蛋白的构象(S)的长期问题将揭示变构 突变改变了它，它将阐明什么αβ-微管蛋白构象可以被TOG域识别。 我们的方法是独特的，并承诺提供以前无法获得的基本洞察力 MT的动态和调节机制。