【作者】 张海燕；

【导师】 牛立文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 翻译后修饰是对于蛋白质发挥生物学功能必不可少的化学修饰,这些翻译后修饰包括泛素化,SUMO化和甲基化等。α-N末端甲基化是普遍存在的翻译后修饰,在整个原核生物和真核生物中都保守。最初报道的蛋白α-N末端甲基化修饰发生在细菌核糖体蛋白L16和L33上。随后,许多蛋白质如染色质浓缩调节子1(RCC1),CENP-A/B和受损DNA结合蛋白2(DDB2)等都经历了 α-N端甲基化修饰。总体而言,α-N末端甲基化涉及多种细胞过程,如有丝分裂,发育进程和DNA损伤修复。ScNTM1是酿酒酵母(Saccharomyces cerevisiae)中YBR261C/Tae1基因编码的α-N末端甲基转移酶(本文称之为ScNTM1),可将核糖体蛋白Rpl12ab和Rps25a/Rps25b甲基化。同时,该酶也是首次报道的能将真核生物核糖体蛋白α-N末端甲基化修饰的酶。基因敲除研究表明,甲基化的核糖体蛋白可能保护酵母免受微生物侵袭并且对于蛋白质的合成有重要作用。然而,对来自酵母的ScNTM1的生化及其与底物相互作用的分子机制方面的研究却鲜有报道。为了阐明ScNTM1与其底物之间的分子相互作用,本文获得了酿酒酵母ScNTM1-SAH-Rps25a衍生的六肽(PPKQQL)的三元复合物以及ScNTM1-SAH的二元复合物的晶体结构。结构分析表明ScNTM1有一个与其他SAM-MTase中同样的用于SAM/SAH结合的核心结构域,蛋白分子表面有些结构发生变化,显示出与进化有关的结构变异。与SAH-NRMT1相比,ScNTM1的核心结构域中为SAH结合提供疏水相互作用的残基大部分是保守的,如Gly73,Phe98,Gly122和Leu145。由于Pro94取代了 NRMT1中相应的Ile,腺嘌呤环偏转约20°,以适应腺嘌呤与Pro94之间的环环堆积作用。通过比较ScNTM1和人源NRMT1,NRMT2的三元复合结构,我们发现参与Rps25a底物结合的残基大多数是保守的。底物的前两位氨基酸与蛋白结合时涉及的关键残基是相同的,如Asn172,Trp140。后四位由于底物多肽的摆动方向不同,因此与蛋白相互作用的氨基酸发生了变化。ScNTM1结构中Q4与Tyr32的侧链的氢键是特有的,该氢键在P2和多肽中的Q4识别中起重要作用。参与Rps25a和SAH结合的关键残基也通过点突变和体外等温滴定量热(ITC)实验进行了验证。ScNTM1的二元和三元复合物结构比对发现多肽结合后某些区域,如残基25-36,183-186,215-221部位发生了轻微的构象变化。这些研究丰富了人们对α-N末端甲基转移酶底物的选择性及特异性的认识。更多还原

【Abstract】 Post-translational modifications refer to chemical modifications that are essential for mature biological function of proteins.Proteins from eukaryotic origins undergo various post-translational modifications i.e.ubiquitination,SUMOylation and methylation.The first reported N-terminal methylation modification of proteins occurred on bacterial ribosomal proteins L16 and L33.Subsequently,many proteins such as regulator of chromatin condensation 1(RCC1),centromere protein A/B(CENPA/B)and damaged DNA-binding protein 2(DDB2)have been reported to undergo Nterminal methylation.Overall,N-terminal methylation involves a wide range of functions in cellular process,i.e.mitotic,development progression and DNA damage repair.ScNTM1 is an α-N terminal methyltransferase encoded by YBR261C/Tae1 gene in Saccharomyces cerevisiae(herein referred to as ScNTM1),which methylates ribosomal proteins Rp112ab and Rps25a/Rps25b.It is also the first reported enzyme that can methylate the α-N terminal of ribosomal protein in eukaryotes.Previous knockout studies demonstrated that the methylated ribosomal proteins might protect yeast from microbial attack and play an important role in protein synthesis.However,few studies have been reported on the biochemistry of ScNTM1 from yeast and its molecular mechanism of interaction with substrates.To elucidate the molecular interactions between the ScNTM1 and its substrate,the crystal structures of ScNTM1 from Saccharomyces cerevisiae in complex with SAH and Rps25a-derived hexapeptide(PPKQQL)as well as the binary complex of ScNTM1-SAH were obtained.Structure analysis revealed that ScNTM1 adopted a core methyltransferase fold the same as other solved SAM-MTases for SAH-binding.Some structural changes on the surface of ScNTM1 display evolution-related variations.Compared to SAH-NRMT1,most of the residues in the core domain of ScNTMl that provide hydrophobic interactions for SAH binding are conserved,such as Gly73,Phe98,Gly122 and Leu 145.As Pro94 replaces the corresponding Ile in NRMT1,the adenine ring deflects about 20°to adapt to the ring-ring stacking between adenine and Pro94.By comparing the ternary complex structure of ScNTM1 and human NRMT1 and NRMT2,we found that most of the residues involved in the binding of Rps25a substrate are conserved.The key residues participating in the binding of the first two amino acids of the substrate to the protein are the same,such as Asn172 and Trp140.The last four amino acids that interact with the protein have changed due to the different swing directions of the substrate polypeptide.The hydrogen bond between Q4 and the side chain of Tyr32 is unique to ScNTM1,and this hydrogen bond plays an important role in the recognition of P2 and Q4 in polypeptides.The key residues involved in the binding of Rps25a and SAH were verified by point mutation and isothermal titration calorimetry(ITC)experiments in vitro.Structural alignment of the ternary and binary complex indicate a slight shift occurring in some regions i.e.residues 25-36,residues 183-186 and residues 215-221 upon peptide binding.These studies have enriched our understanding of the selectivity and specificity of α-N-terminal methyltransferase substrates.更多还原