Mechanism of tubulin modification enzymes

微管是细胞形态发生、细胞分裂和细胞内运输所必需的聚合物。它们受到高度多样化、丰富性和进化保守性的翻译后修饰。微管蛋白修饰水平和模式的破坏会导致癌症、神经病理和轴突再生缺陷。破译微管蛋白密码的一个重要方面是了解密码是如何编写的，即引入这些修饰的酶的机制，以及这些酶之间的合作和竞争如何导致在细胞中观察到的复杂的微管修饰模式。具体地说，我们的目标是(1)确定关键的微管蛋白修饰酶在分离以及与微管的复合体中的高分辨率结构，以了解它们的底物特异性和催化机制；(2)绘制所有修饰酶的微管蛋白修饰位点图；(3)研究微管蛋白修饰酶之间的生化相互作用以及这如何导致时间和空间调节的修饰模式。该项目利用我们制造未经修饰和重组的单一异构体工程化人类微管蛋白的能力，并结合我们的一系列结构技术(X射线结晶学、冷冻EM和SAXS)、高分辨率质谱学、经典动力学和单分子荧光，将回答有关微管蛋白修饰酶的机制和调控的基本问题。 去年，我们在实现这些目标方面取得了重大进展。具体地说，我们专注于TTLL谷氨酸酶，这是微管蛋白修饰酶中最大的家族。谷氨酰化是翻译后将谷氨酸链加到微管蛋白C末端的过程。这是人脑中最丰富的微管蛋白修饰。运动和微管相关蛋白对短谷氨酸链和长谷氨酸链的反应不同。与泛素密码类似，TTLL谷氨酸酶被认为是专门化的起始酶，它从微管蛋白尾部的内部谷氨酸(通过异肽键)分支多聚谷氨酸链，并从分支点延长长链，无论是线性的还是分支的。结合MS-MS和核磁共振，我们应该认为谷氨酰基酶TTLL6优先延长α-微管蛋白微管蛋白尾部的线性谷氨酸链，并且这种活性随着延伸支链中谷氨酸数量的增加而增加，这与较强的延长活性一致。相反，谷氨酰胺酶TTLL4不会延长链，而是在β-微管蛋白尾部的多个内部位置引入单谷氨酸分支(Mahalingan等人。自然结构Molec Biol 2020)。 此外，我们还确定了TTLL6的几种基于机制的抑制剂。TTLL6与这些抑制剂的复合体的共晶结构与蛋白质工程相结合，揭示了关键的活性部位残基，这些残基控制着酶是延长还是只是启动线性谷氨酸链。这些残基的突变将TTLL6从伸长酶转换为起始酶，表明这些残基是必要的，并且足以指定区域选择性(Mahalingan等人。自然结构Molec Biol 2020)。这些残基与TTLL家族中的起始酶和延伸酶分离。因此，我们的结构和功能工作为所有TTLL酶的区域选择性奠定了分子基础。 因此，我们在过去一年的工作(1)阐明了TTLL起始酶和延伸酶的活性位点签名，并严格证明了这些机制的系统发育保守；(2)它揭示了在细胞中产生组合微管复杂性的基本原理；(3)它增加了我们用于生成差异修饰微管的工具箱，用于进一步分析微管编码；(4)我们的结构使未来TLL活性位点的合理工程能够与氨基酸类似物兼容，类似于为使对其功能的研究产生革命性研究的激酶所做的补偿性突变。由于无法快速抑制或激活修饰酶，以及缺乏用于活细胞成像的微管蛋白修饰报告，阐明微管蛋白密码如何调节细胞生理的进展仍然受到阻碍；(5)由于TTLL酶的谷氨酰化作用作为一种广泛蛋白质的调节因子，包括组蛋白伴侣蛋白、c-GAS酶和多能转录因子，我们的研究提供了一个机制框架来了解它们与非微管蛋白靶点的相互作用，并将作为设计抑制剂的跳板，用于治疗以微管蛋白高谷氨酸氨化或cGAS谷氨酸氨化调节固有免疫反应为特征的神经退行性疾病的治疗。

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved posttranslational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. An essential aspect of deciphering the tubulin code is to understand how the code is written i.e. the mechanism of the enzymes that introduce these modifications and how cooperation and competition between these enzymes gives rise to the complex microtubule modification patterns observed in cells. Specifically we aim (1) to determine high-resolution structures of key tubulin modification enzymes in isolation as well as in complex with the microtubule to understand their substrate specificity and catalytic mechanism; (2) to map tubulin modification sites for all modification enzymes; (3) to investigate the biochemical interplay between tubulin modification enzymes and how this gives rise to temporally and spatially regulated modification patterns. This project leverages our ability to make unmodified and recombinant single-isoform engineered human tubulin and coupled with our expertise with an array of structural techniques (X-ray crystallography, cryo-EM and SAXS), high-resolution mass spectrometry, classical kinetics and single molecule fluorescence will answer fundamental questions about the mechanism and regulation of tubulin modification enzymes. We have made significant progress towards these goals in the last year. Specifically, we focused on TTLL glutamylases, the largest family of tubulin modification enzymes. Glutamylation is the post translational ATP-addition of glutamate chains to the tubulin C-terminal tails. It is the most abundant tubulin modification in the human brain. Motors and microtubule associated proteins respond differently to short versus long glutamate chains. Analogous to the ubiquitin code, TTLL glutamylases are thought to specialize as initiatiases, that branch the polyglutamate chain from an internal glutamate in the tubulin tail (through an isopeptide bond), and elongates, those that elongate long chains, either linear or branched, from the branch point. Combining MS-MS and NMR we should that the glutamylase TTLL6 preferentially elongates linear glutamate chains on the tubulin tails of alpha-tubulin and that this activity increases with the number of glutamates in the extended branch, consistent with strong elongation activity. In contrast, the glutamylase TTLL4 does not elongate chains, but introduces monoglutamate branches at multiple internal positions in the beta-tubulin tail (Mahalingan et al. Nature Struct Molec Biol 2020). Moreover, we identified several mechanism-based inhibitors for TTLL6. Co-crystal structures of TTLL6 in complex with these inhbitors coupled with protein engineering revealed key active site residues that control whether the enzyme elongates linear glutamate chains or just initiates them. Mutation of these residues switches TTLL6 from an elongase to an initiase, indicating that these residues are necessary and sufficient to specify regioselectivity (Mahalingan et al. Nature Struct Molec Biol 2020). These residues segregate with initiases and elongases across the TTLL family. Thus, our structural and functional work lays bare the molecular basis for regioselectivity for all TTLL enzymes. Thus our work in the past year (1) elucidates the active site signatures for TTLL initiases and elongases and rigorously demonstrates the phylogenetic conservation of these mechanisms; (2) it uncovers the underlying principles for generating the combinatorial tubulin complexity in cells; (3) it adds to our toolbox for generating differentially modified microtubules for further analyses of the tubulin code; (4) Our structures enable the future rational engineering of TTLL active sites to be compatible with amino acid analogs, similar to the compensatory mutations made for kinases that have revolutionized studies into their function. Progress in elucidating how the tubulin code regulates cell physiology continues to be hampered by the inability to rapidly inhibit or activate modification enzymes and a lack of tubulin modification reporters for live cell imaging;(5) Since glutamylation by TTLL enzymes functions as a regulator of a wide range of proteins including histone chaperones, c-GAS enzymes, and pluripotency transcription factors, our study provides a mechanistic framework to understand their interactions with non-tubulin targets and will serve as a springboard for the design of inhibitors for therapeutic intervention in neurodegenerative disorders characterized by tubulin hyper-glutamylation or modulation of the innate immune response by cGAS glutamylation.