人类GMP还原酶2（GMPR2）的结构与功能研究

【作者】 李继喜；

【导师】 谢毅；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 嘌呤代谢是核苷酸代谢的核心过程，广泛参与细胞的生理生化过程，对于生物体的生长、发育、分化、增殖以及凋亡至关重要。GMP还原酶（GMPR，EC1.7.1.7）在辅酶NADPH的作用下催化GMP不可逆地脱氨生成IMP，并在重新利用胞内自由碱基和嘌呤核苷，维持腺嘌呤和鸟嘌呤核苷酸的平衡方面起着重要的作用。但目前对GMPR的作用机理、催化机制以及相关功能的研究还不清楚。本研究成功建立了人类GMPR2原核表达纯化系统，获得了高纯度（＞95％）的融合蛋白，并对其结构和功能进行了研究。采用汽相悬滴扩散法在4℃条件下培养出GMPR2与底物GMP的蛋白质晶体，收集和分析了该晶体的X-射线衍射数据，衍射分辨率为3.0 （?）。GMPR2晶体的空间群属于P3₂21晶系，晶胞参数分别为a=b=110.6 （?），c=209.8 （?）（α=β=90°，γ=120°）。以Thermotoga Maritima IMP脱氢酶（PDB ID：1VRD）的晶体结构为模型，采用分子置换法解析出GMP还原酶家族第一个成员，即人类GMPR2与GMP复合物的晶体结构（PDB ID：2A7R）。人类GMPR2晶体结构为四个亚基组成的同源四聚物，每个单体由11个α螺旋和14个β折叠组成，并且在核心位置都形成一个（a／β）₈桶状结构。有趣的是，底物GMP通过与氨基酸残基Met269、Ser270、Arg286、Ser288和Gly290发生相互作用来结合GMPR2；这就使得GMP临近的柔性结合区域（残基268-289）的构像类似于一个可以转动的铰链门——GMP结合紧密则该区域构像闭合，不然则处于无序状态。与GMPR1和IMP脱氢酶家族的结构同源性比较表明它们的活化位点环（残基179-187）的构像相似。我们推测Cys186可能是GMPR2的活性位点，而残基129-133所在的环对结合NADPH起作用。该蛋白的晶体结构解析对于理解GMPR家族的功能活性起着重要的作用。根据GMPR2的晶体结构分析，我们对一些关键氨基酸残基进行了定点突变实验以研究它们的生化效应。实验结果表明Cys186和Thr188的突变对GMPR2影响很大，几乎使GMPR2的活性完全丧失；而Ser184和Asn132的突变会引起GMPR2的活性部分丧失。我们又对GMPR2的生物学功能进行了研究。逆转录PCR（RT-PCR）显示，GMPR2在人体多种组织及肝癌、肺癌、胃癌等组织中广泛表达。对基因芯片表达谱数据进行聚类分析，发现GMPR2的相关基因中包括APRIN、IFIT1等细胞增殖相关基因。绿荧光融合蛋白表达显示GMPR2在不同细胞中分布有所不同，COS-7细胞中定位于细胞质，而在AD293细胞中定位于细胞质和细胞核。用RNAi方法来抑制GMPR2在细胞内的表达，结果显示对细胞增殖水平（非沉默RNA做为对照：22.76±1.43％；三对SiRNA中GMPR2-1：25.12±1.84％；GMPR2-2：26.72±1.24％；GMPR2-3：21.34±1.72％）和细胞凋亡未见显著影响。但在AD293细胞中发现过表达GMPR2可以引起细胞增殖水平上升（对照：37.12±1.28％；GMPR2：58.36±1.53％），而对细胞凋亡无显著影响。本论文对GMPR2的晶体结构和生物学功能进行了研究。通过X—射线衍射法，我们首次得到并解析了GMPR家族成员之一，人类GMPR2与GMP复合物的晶体结构（PDB ID：2A7R），分析了GMPR2的三维结构特点及可能的催化作用位点；通过关键位置氨基酸的定点突变研究了它们的生化效应，并对相关的生物学功能（组织表达谱、芯片研究、亚细胞定位、RNAi及真核细胞过表达等）进行了研究。更多还原

【Abstract】 Purine metabolism is essential in nucleoside metabolism, involving in the biochemical and physiological process, and plays key roles in cell growth, development, differentiation, proliferation, and cell apopotosis. Guanosine monophosphate reductase （GMPR, EC 1.7.1.7） catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in reutilization of free intracellular bases and purine nucleosides and maintaining the balance of Adenosine and Guanosine in cell. However, it is unclear for GMPR about the reaction mechanism and functional research so far.In this study, GMPR2 fusion protein was expressed successfully in E.Coli cells and purified with 95% homogenicity. Complex of GMPR2 and substrate GMP was crystallized by using the method of hanging-drop vapor-diffusion at 4℃. X-ray diffraction analysis revealed that the crystals belong to the space group P3₂21 with unit-cell parameters a = b = 110.6 （?）, c = 209.8 （?） （a=β=90°, γ =120°） , and they diffracted to at least 3.0 A resolution. The first crystal structure of GMPR family （Protein Data Bank entry 2A7R） was solved through the molecular replacement method using the 2.18 A resolution structure of Thermotoga Maritima IMP dehydrogenase as a search model （PDB entry 1VRD）.The GMPR2 formed a tetramer composed of subunits adopting the ubiquitous （a/β）₈ barrel fold. Each subunit was comprised of 11a and 14β. Interestingly, the substrate GMP was bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this made the conformation of the adjacent flexible binding region （residues 268-289） fixed, much like door on a hinge. Structures comparison and sequence alignment analyses showed that the conformation of the active site loop （residues 179-187） was similar to those of hGMPR1 and Inosine Monophosphate Dehydrogenases （IMPDHs）. We proposed that Cys186 was the potential active site, and that the conformation of the loop （residues 129-133） suggested a preference for the coenzyme NADPH over NADH. This structureprovides important information towards understanding the functions of members of the GMPR family.Site-directed mutations of several conserved amino acids were used to understand their characterizations according to the structural analysis of GMPR2. The results showed that Cysl86Ala and Thr188Ala abolished hardly the enzyme activity of GMPR2; Serl84Ala and Asnl32Ala caused partly inactivation related to the wild type of GMPR2. Reverse transcription-PCR analysis showed that GMPR2 was expressed ubiquitously in human tissues, liver adenocarcinoma, lung adenocarcinoma, and stomach adenocarcinoma tissues. Cluster analysis of microarray data revealed some GMPR-related genes, including APRIN and IFIT1, involved in cell proliferation. Subcellular location with green fluorescent protein （GFP） showed that GMPR2 was distributed in the cytoplama in COS-7 cells, while expressed both in cytoplama and nucleus in AD293 cells. The expression of GMPR2 was silenced by RNA interference （RNAi） method. The results showed it did little effection to the level of cell cycle （non-silencing RNA: 22.76±1.43%; within the three siRNA, GMPR2-1: 25.12±1.84 % ;GMPR2-2: 26.72±1.24 % ; GMPR2-3: 21.34±1.72 % ） and cell apopotosis. Overexpression of GMPR2 in AD293 cells resulted in the elevated level of cell proliferation （control: 37.12±1.28%; GMPR2: 58.36±1.53%）, but no effect on cell apopotosis.The structural and functional researches of human GMPR2 were performed in this paper. We successfully resovled the first crystal structure of GMPR family （Protein Data Bank entry 2A7R） and analyzed its characterization and active site. Moreover, mutants, expressed profile in different tissues, subcellular location, and RNAi researches were used to study the function of human GMPR2.更多还原