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，丰富和进化保守的翻译后修饰。微管蛋白修饰水平和模式的破坏导致癌症、神经病变和轴突再生缺陷。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引起细胞中观察到的复杂的微管修饰模式。具体来说，我们的目标是：(1)确定关键微管蛋白修饰酶的高分辨率结构，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；(3)研究微管蛋白修饰酶之间的生化相互作用，以及这种相互作用如何引起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异构体工程人微管蛋白的能力，并结合我们的专业知识与一系列结构技术（x射线晶体学，低温电镜和SAXS），高分辨率质谱，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节的基本问题。

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.