蛋白质N末端甲基转移酶的结构及催化机制研究

Protein N-terminal methylation is a unique post-translational modification that plays essential roles in mitosis and protein synthesis. Protein N-terminal methylation is catalyzed by protein N-terminal methyltransferases. So far, only three N-terminal methyltransferases have been identified in human, referred to as NTMT1, NTMT2 and METTL13. We previously determined the crystal structures of NTMT1/2 in complex with substrates and SAH that have shed light on their catalytic mechanisms. The novel N-terminal methyltransferase METTL13 is recently discovered in human, its function homolog denoted Efm7 in yeast. METTL13-catalyzed eEF1A methylation enhances protein synthesis and promotes pancreatic and lung tumorigenesis. However, the molecular mechanism of methylation of eEF1A by METTL13 remains elusive. In order to unveil the molecular basis of substrate recognition and catalysis by METTL13/Efm7, we intend to employ X-ray crystallography to determinate the crystal structures of METTL13/Efm7 in ternary complex with substrate and cofactor. Structural observations combined with biochemical studies will reveal the residues that responsible for substrate recognition and catalytic activity, and delineate the catalytic mechanism of METTL13/Efm7. Our results will be crucial for the further understanding of the molecular mechanism behind METTL13-catalyzed eEF1A methylation, and also provide structural basis for the design of small-molecule inhibitors as well as facilitate the discovery and development of new anticancer drugs.

蛋白质N末端的甲基化是一类独特的翻译后修饰，在细胞分裂和蛋白质合成中发挥着重要的作用。目前在人源中只鉴定出三个能够催化这种修饰的蛋白质N末端甲基转移酶：NTMT1、NTMT2和METTL13。申请人之前的研究阐述了NTMT1和NTMT2的催化机制。最新鉴定的METTL13能够甲基化eEF1A，从而加速蛋白质的合成，促进胰腺癌和肺癌的发生。然而METTL13的催化机制还并不清楚。本项目拟利用结构生物学解析METTL13和其酵母中的同源蛋白Efm7分别与底物及甲基供体的三元复合物结构，并结合生物化学方法鉴定底物识别和酶活催化的关键位点，阐明它们的具体催化机制。本项目的研究结果对于深入理解METTL13介导的eEF1A甲基化调控蛋白质翻译的机制具有重要意义，也为进一步设计和筛选小分子抑制剂提供结构基础，并最终为抗癌药物的研发提供理论指导意义。

蛋白质N末端的甲基化是一类独特的蛋白质翻译后修饰，在原核生物和真核生物中普遍存在。这种修饰是通过蛋白质N末端甲基转移酶来催化完成的。蛋白质N末端甲基转移酶的不稳定调控与DNA修复缺陷、早衰、不育、癌症发生等密切相关。因此，对蛋白质N末端甲基转移酶催化机制的研究不仅有助于深入了解N末端甲基化的生理功能，也能够促进更多生理底物的鉴定，为进一步化学小分子抑制剂和临床药物的研发提供理论基础。本项目通过X-射线晶体学和生物化学等技术深入研究了METTL13和Efm7的结构和功能，揭示了两者底物识别的特异性和酶活的催化机制。在此基础上，进一步阐明了CDY家族蛋白及PHF1和PHF19识别组蛋白的甲基化修饰的分子机制。跟据CDY底物识别位点，设计合成了一个化学小分子抑制剂，能够特异性结合CDYL1/2，并解析了此小分子和CDYL2的复合物结构，阐明了此小分子对CDYL2的抑制机制。本项目的研究完善了人们对蛋白质甲基转移酶催化机制以及METTL13介导的甲基化调控蛋白质翻译机制的理解，而且进一步完善了不同甲基化组蛋白被识别的分子机制，为进一步设计并筛选小分子抑制剂调节METTL13及甲基化识别蛋白的功能提供分子基础，并最终为癌症的治疗提供理论指导意义。