Molecular dissection of the C-terminal tails of tubulin and the effect of their polyglycylation on binding and microtubule assembly

首席调查员/项目主任(最后、第一、中间)：Hough，Loren E. 由↵-微管蛋白杂二聚体制成的微管对细胞迁移、长距离运输、 和细胞分裂。微管蛋白调节的一个主要部位是C末端尾巴(CTTS)。主要问题仍然存在 关于CTT功能和调控的分子机制。CTT影响微管长度动力学和 机械性能，即使它们只贡献了结合界面的一小部分 相邻的二聚体。CTT是微管蛋白翻译后修饰fi阳离子的一个主要部位，它调节微管蛋白的 绑定相互作用。CTTS的pTm改变了运动蛋白1、2的加工性和蛋白质的affi刚性，从而 影响MT稳定性(例如MCAK、CLIP-170)3、4和稳定MT的蛋白质(例如tau)。5 尽管它们很重要，但关于CTT行为的分子探针很少。因为CTT是fl可执行的，所以它们 在电子显微镜或X射线结晶学研究中通常是检测不到的。核磁共振是最好的方法 CTT调节MT聚合的分子机制、力学、结合和调节 通过PTMS。然而，掺入重同位素的标准技术对微管蛋白并不起作用。 蛋白质还没有在原核系统中合成，而微管蛋白通常下调自己的表达， 在真核系统中实现fi的高效表达。我们开发了一种生产重同位素标记的方法 微管蛋白用于核磁共振研究，从而可以详细研究CTT功能的分子机制。 在这一突破的基础上，我们将研究CTT多甘氨酸化在MT调节中的作用。增加了 甘氨酸残基到谷氨酸侧链存在于两个微管蛋白CTTS上。First Identifi作为两大 微管蛋白上的多聚-莫迪fi阳离子，多甘氨酸与特别稳定的MT有关，特别是轴丝 在活动纤毛中。6减少聚甘氨酸酯化的突变与纤毛的稳定性降低有关，减少 纤毛数量，细胞增殖增加，以及癌症。然而，目前尚不清楚为什么聚甘氨酸会导致 这些影响。PTMS可以通过多种机制工作：Modifi阳离子的整体电荷分布，7 结合界面的改变，8通过变构机制诱导结构变化，9和调制 我们将结合核磁共振、结合分析、分子模拟和细胞生物学 检测CTT功能的可能机制。我们将重点研究两个问题： 1.多聚甘氨酸对MT聚合动力学和刚性有何影响？CTT的存在 影响MT聚合动力学和力学性能，表明一个二聚体的CTTS相互作用 与MT格子中的其他二聚体一起。11，12我们将纯化重同位素标记的微管蛋白 使用TTLL3家族蛋白突变和微管蛋白CTT突变的多甘氨酸化。 我们将使用MT组装试验来确定聚甘氨酸对MT成核和聚合的影响 动力学。我们将通过对二聚体和聚合物的核磁共振研究聚甘氨酸对CTT性质的影响。 简化的MTS。结合分子模拟，我们将确定并重新fi分子的Ne假设。 CTT多甘氨酸化影响MT特性的机制。我们将通过突变来检验这些假说。 2.多聚甘氨酸如何影响MT结合作用？去除CTTS改变了粘合模具- MT-稳定和破坏结合伙伴的动态，提示CTT PTM可能通过 对MT相互作用蛋白的影响。然而，关于MT结合伙伴的结构信息很少 识别CTT，即使是那些交互依赖于PTM的CTT。例如，CTTS的PTM改变了 运动蛋白1，2的加工性和影响MT稳定性的蛋白质的affi(如Mca，CLIP-170)。3，4 我们将确定MT结合蛋白的fi结合强度如何随CTT多聚甘氨酸的程度而变化。 提顿。通过核磁共振，我们将确定CTT与MT结合蛋白的结合界面，并确定 当多聚甘氨酸化程度改变时，结合界面是否改变以及如何改变。我们将决定 结合后对环境影响最大的残留物。这项工作将使我们能够生成和测试假设 关于聚-G尾巴影响MT结合特性的分子机制。 回答这些问题将为微管蛋白CTT翻译后修饰fi阳离子提供独特的见解 调节MTS。我们做这项工作的准备是独一无二的，因为我们有CTT平均值的直接读数 通过使用核磁共振环境和绑定接口。这个项目将是一个详细的表征 微管蛋白CTT多聚甘氨酸调节细胞MT的机制及为解剖提供框架 无序结构域的PTM对细胞蛋白质的调节。在微管蛋白物种fiCally之外，嗜热T 大量的标记、过度表达和分泌系统将使我们的方法能够进行核磁共振结构研究 种类繁多的真核生物蛋白质。 计划(日期)第0页建议书文本

Principal Investigator/Program Director(Last, First, Middle): Hough, Loren E. Microtubules (MTs) made from ↵- tubulin heterodimers are important for cell migration, long range transport, and cell division. A primary site of tubulin regulation is the C-terminal tails (CTTs). Major questions remain about the molecular mechanism of CTT function and regulation. CTTs affect microtubule length dynamics and mechanical properties even though they contribute only a small percentage of the binding interface between adjacent dimers. CTTs are a major site of tubulin post-translational modification (PTM) which regulates tubulin's binding interactions. PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which affect MT stability (e.g. MCAK, CLIP-170)3, 4 and proteins which stabilize MTs (e.g. tau).5 Despite their importance, there are few molecular probes of CTT behavior. Because CTTs are flexible, they are typically undetectable in electron microscopy or x-ray crystallography studies. NMR is the best approach for determining the molecular mechanism of CTT regulation of MT polymerization, mechanics, binding and regulation by PTMs. However, standard techniques for incorporation of heavy isotopes have not worked for tubulin—the proteins have not yet been made in prokaryotic systems, and tubulin typically down regulates its own expression, making over-expression difficult in eukaryotic systems. We developed a method to produce heavy isotope labeled tubulin for study by NMR, allowing for a detailed study of the molecular mechanism of CTT function. Building on this breakthrough, we will study the role of CTT polyglycylation in MT regulation. The addition of glycine residues to glutamate side chains occurs on both tubulin CTTs. First identified as one of the two major poly-modifications on tubulin, polyglycylation is associated with particularly stable MTs, especially axonemes in mobile cilia.6 Mutations that decrease polyglycylation are associated with reduced stability of cilia, reduced numbers of cilia, increases in cell proliferation, and cancer. However, it is not known why polyglycylation causes these effects. PTMs can operate through a variety of mechanisms: modification of the overall charge distribution,7 alteration of binding interfaces,8 induction of structural changes through allosteric mechanisms,9 and modulation of disordered protein ensembles.10 We will combine NMR, binding assays, molecular simulation, and cell biology to test potential mechanisms of CTT function. We will study two focused questions: 1. How does polyglycylation affect MT polymerization dynamics and stiffness? The presence of the CTT affects MT polymerization dynamics and mechanical properties, suggesting that the CTTs of one dimer interact with other dimers in the MT lattice.11,12 We will purify heavy isotope-labeled tubulin with varying degrees of polyglycylation using mutations in TTLL3-family proteins and mutations in the tubulin CTT at polyglycylation sites. We will use MT assembly assays to determine the effect of polyglycylation on MT nucleation and polymerization dynamics. We will study the effects of polyglycylation on CTT properties by NMR for both dimers and polymer- ized MTs. In conjunction with molecular simulations, we will determine and refine hypotheses for the molecular mechanisms by which CTT polyglycylation affects MT properties. We will test these hypotheses through mutation. 2. How does polyglycylation affect MT binding interactions? Removal of the CTTs alters the binding dy- namics of MT-stabilizing and destabilizing binding partners, suggesting that CTT PTM may regulate MTs through effects on MT-interacting proteins. However, little structural information is available on how MT binding partners recognize the CTTs, even those whose interaction is PTM dependent. For example, PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which affect MT stability (e.g. MCAK, CLIP-170).3, 4 We will determine how the binding affinity of MT-binding proteins varies with the degree of CTT polyglycyla- tion. By NMR, we will determine the binding interface between the CTT and MT-binding protein, and determine whether and how the binding interface changes when the extent of polyglycylation is altered. We will determine the residues most affected in environment upon binding. This work will allow us to generate and test hypotheses about the molecular mechanism by which poly-G tails affect MT-binding properties. Answering these questions will give unique insight into how tubulin CTT post-translational modification regulates MTs. We are uniquely poised to do this work because we have a direct read-out of CTT average environment and binding interface through the use of NMR. This project will be a detailed characterization of the mechanisms of cellular MT regulation by tubulin CTT polyglycylation and provide a framework for dissecting cellular protein regulation through PTM of disordered domains. Beyond tubulin specifically, the T. thermophila heavy labeling, overexpression, and secretion systems will allow our approach to enable NMR structural studies of a wide variety of eukaryotic proteins. Program (Date) Page 0 Proposal Text