含锌指tRNA修饰酶TiaS催化活性的结构基础与远程影响机制研究

Archaeal TiaS (tRNAIle2 agmatidine synthetase) protein is a four domain tRNAIle2 modifying enzyme, which is able to hydrolyze ATP and modify the 2’carbon of the wobble position cytosine 34 with agmatine(Agm). The modification is essential for tRNAIle2 maturation and thus for accurate deciphering of AUA codon. The enzyme core consisting of the N terminal three domains is responsible for the catalytic activity. With a distance of more than 60Å, the C-terminal zinc ribbon domain (ZRD) is far away from the catalytic centre. Residues Glu360 and Arg369 of ZRD are both involved in substrate tRNAIle2 binding, and the distance between the two is about 5Å. R369A mutant shows decreased activity of tRNAIle2 modification, while E360A point mutation increases catalytic activity by about 3 fold. Then the agmatidine formation activity of E360K is examined by the applicant, and it’s found to be decreased. This phenomenon is distinct from either allosteric regulation, or effect of regulatory subunit on catalytic subunit observed in enzyme complexes, both of which take effect through modulating the catalytic centre. Does this mean that the 360 residue ‘remote controls’ the catalytic activity of the enzyme without affecting the enzyme catalytic core? This intriguing phenomenon uncovers a previous unknown critical aspect of enzymology and biochemistry at molecular level, of how long-range effect on catalysis being achieved within a biological macromolecular enzyme. Based on previous work we published on TiaS, we plan to pursue this question by performing structural, biochemical and biophysical research into wild type and series of TiaS-E360 mutants, ① to determine their crystal structures, ②to examine their ATP hydrolysis activity and ③substrate tRNAIle2 modification activity (kon, koff, kcat, KM, kcat/KM), ④to examine the interaction (including affinity KD) between substrate tRNAIle2, ATP-Mg, agmatine and these proteins by ITC or SPR. For TiaS mutagenesis, Glu360 will be replaced by residues with side chains of different size, charge, polarity and hydrophobicity etc. Careful analysis of these results will reveal the inner mechanism of the impact of TiaS-E360 mutation on catalytic efficiency of modification. To summarize, this research will enrich our knowledge about biological macromolecular enzymes and provide significant insight into long-range-effect mechanism of catalysis. This research would also pave ways for enzyme engineering, catalytic centre alteration might not be necessarily required to achieve enzyme function manipulation, but remote modification could be an option instead. Zinc finger engineering and targetable nucleases (using zinc fingers or CRISPR/Cas) engineering have wide applications ranging from research to medicine. New characteristics about zinc ribbon and zinc ribbon-nucleic acid interaction discovered from this study may help future designs of targetable proteins for applications in genetic modulation, genome manipulation or cancer therapeutics research.

TiaS蛋白水解ATP，然后用胍基丁胺agmatine修饰底物tRNA使其成熟。远离酶活性中心超60Å的锌指结构域，其改变可上下调节酶活性！锌指的E360、R369之间距离约5Å，均参与了tRNA的结合。突变体R369A、E360K修饰活性显著下降，但E360A酶活大幅上升。酶的催化活性是否、如何进行远程调控，目前并无相关理论。此独特现象揭开了生化领域的一个重要新命题。在前期工作基础上，本项目拟对野生型与系列E360突变体(改变残基大小、电荷、亲疏水性等)开展晶体学、生物物理学(ITC/SPR)、酶学等研究，测定并比较它们的①结构、②ATP水解活性、③tRNA修饰活性（kon、koff、kcat、KM、kcat/KM）、④与ATP-Mg, agmatine, tRNA相互作用及亲和力KD的相似与不同之处，对单氨基酸长距离影响酶活的因素与分子机制进行深入探索，为分子水平解读生物大分子酶催化机理开辟新思路。本研究对酶及锌指改造的应用研究也具有重要意义。

TiaS蛋白的远离酶活性中心超60Å的锌指结构域，其改变可上下调节酶活性！锌指的突变体R369A修饰活性显著下降，但E360A酶活大幅上升。酶的催化活性是否可以、如何进行远程调控?此独特现象揭开了生化领域的一个重要新命题。.本项目首先在理论上，分析了酶催化过程与动力学机制，阐述了酶的催化活性被远离活性中心的因素进行远程调控在理论上是符合逻辑、符合生化基本规律的。影响催化过程中任何一个步骤的因素都可能影响催化速率，不论是远程的还是在催化活性中心的。酶的催化活性如何进行远程调控？围绕此问题开展了实验。.①对系列锌指结构域突变体蛋白(改变关键残基大小、电荷、极性、亲疏水性等)开展了晶体学研究，测定并比较了它们的结构与构象。使用SAD，比较了野生型、突变体锌指结构域络合金属原子的情况，获得了含有以及不含有X-射线反常散射信号的结构。.②解析TiaS-E360突变体蛋白与辅因子复合物高分辨率晶体结构。研究了野生型、突变体与ATP-Mg、agmatine这些辅因子相互作用的相似与不同之处。.③通过小角散射实验Bio-SAXS获得溶液中野生型、E360突变体蛋白的外形与构象，并与晶体结构中的结构进行比较分析。.④构建E360突变体蛋白TiaS-tRNAIle2起始结合复合物结构模型，使用计算结构生物学技术，进行分子动力学模拟研究，对野生型、突变体的催化机制进行了分析。另使用常规模态分析技术，对野生型、突变体的运动模式的相似与不同之处进行分析。.⑤通过结构研究等，以实验支持了嗜热蛋白在高温下维持结构的若干策略。.本项目分析突变体之间以及与野生型相比，与辅因子相互作用、催化过程、催化动力学、催化机制的异同。综合各突变体的酶活、构象、底物亲和力等数据，将分析总结锌指结构域关键氨基酸残基的相关特性长距离影响酶催化活性(ATP水解、tRNAIle2修饰)、如何影响的实验结果，精准描述关键的远程因素。获得并发表了被同行认可的生化过程动力学机制、生化过程基本规律分析。通过本研究，丰富并支持了生物大分子酶的催化机制、酶促动力学基本规律的理论研究。