CONFORMATION & RECOGNITION IN MICROTUBLE DYNAMICS

结构

• 批准号: 10225380
• 负责人: Luke W Rice
• 金额: $ 34.49万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225380
• 关键词: Adopted; Affect; Affinity; Behavior; Binding; Biochemical; Biochemistry; Cells; Chromosome Segregation; Complement; Cryoelectron Microscopy; Data; Family; Genetic Materials; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Individual; Induced Mutation; Lead; Link; Measurement; Mediating; Microtubule Polymerization; Microtubules; Modeling; Molecular; Molecular Conformation; Mutation; Outcome; Paclitaxel; Pharmaceutical Preparations; Polymerase; Polymers; Property; Recombinants; Regulation; Research; Resolution; Site; Specific qualifier value; Structural Biochemistry; Structure; Tubulin; Tubulin Interaction; Vinca Alkaloids; Work; Yeasts; alpha Tubulin; anti-cancer; base; beta Tubulin; design; expectation; genetic regulatory protein; insight; mutant; polymerization; reconstitution; response; segregation

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. The dynamic properties of MTs are central to their function, and they derive from the structural and biochemical properties of individual tubulin subunits and how they interact within the MT lattice. It is increasingly appreciated that tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to control MT elongation, stability, and switching. The long-term goal of this research is to build a structural understanding of how allostery and the tubulin conformation cycle dictate MT dynamics, and of the mechanisms by which regulatory factors control MT dynamics. In prior project periods, we pioneered a powerful approach based on structure-inspired site-directed αβ-tubulin mutants. In the present proposal, through three specific aims, we will build on these themes to provide unique and fundamental new insights into the physical origins and regulatory mechanism of MT dynamics. We will use biochemistry, reconstitution, and modeling to define general biochemical mechanisms for XMAP215-family polymerase activity and processivity. We will reveal through structures how an `allosteric' mutation that alters MT dynamics affects tubulin conformation in human and yeast MTs, and we will provide new conformation cycle mutants to expand our understanding of allostery in MT dynamics. Finally, we will identify biochemical and structural design principles underlying how CLASP TOG interactions with tubulin suppress catastrophe and promote rescue. This work will provide new information about the conformation(s) of αβ-tubulin and how `allosteric' mutations can perturb MT dynamics and tubulin conformation. The work will also expand our understanding of how different TOG domains achieve different regulatory outcomes, with implications for the underlying mechanisms of microtubule dynamics.

微管 (MT) 是染色体分离和细胞内必需的动态聚合物 组织，并且是紫杉醇和长春花生物碱等抗癌化疗药物的直接目标。这 MT 的动态特性对其功能至关重要，它们源自结构和生物化学 单个微管蛋白亚基的特性以及它们如何在 MT 晶格内相互作用。它越来越 认识到微管蛋白亚基采用不同的构象作为 GTPase 依赖性的一部分 聚合动力学，并且调节蛋白选择性地识别这些构象的子集 控制 MT 伸长、稳定性和转换。本研究的长期目标是建立一个结构性的 了解变构和微管蛋白构象循环如何决定 MT 动力学，以及 调节因素控制 MT 动态的机制。在之前的项目期间，我们开创了 基于结构启发的定点 αβ-微管蛋白突变体的强大方法。在本提案中， 通过三个具体目标，我们将在这些主题的基础上提供独特且基本的新见解 探究 MT 动力学的物理起源和调节机制。我们将使用生物化学、重组、 和建模来定义 XMAP215 家族聚合酶活性的一般生化机制 持续性。我们将通过结构揭示改变 MT 动力学的“变构”突变如何影响 人类和酵母MT中的微管蛋白构象，我们将提供新的构象循环突变体来扩展 我们对 MT 动力学变构的理解。最后，我们将确定生化和结构设计 CLASP TOG 与微管蛋白相互作用的基本原则如何抑制灾难并促进救援。 这项工作将提供有关 αβ-微管蛋白构象以及“变构”突变如何的新信息 可以扰乱 MT 动力学和微管蛋白构象。这项工作还将扩大我们对如何 不同的 TOG 领域实现不同的监管结果，并对潜在的影响 微管动力学机制。