【作者】 张诚；

【导师】 龚为民；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2008， 博士

【摘要】 1.人源异戊二烯焦磷酸异构酶的结构生物学研究人源异戊二烯焦磷酸异构酶（IPP异构酶）是异戊二烯类化合物生物合成途径中的关键性的酶,它催化异戊二烯焦磷酸（IPP）与二甲基烯丙基焦磷酸（DMAPP）之间的相互异构转化,而异戊二烯焦磷酸和二甲基烯丙基焦磷酸则是数目众多的异戊二烯类化合物的共同前体。这里我们首先用单波长反常散射的办法解析了人源IPP异构酶1.6（?）的晶体结构,其空间群为P212121,并随后得到了另外两种不同晶型的晶体结构,空间群分别是C2和P1,其中空间群P1的晶体中是无机培养基表达的硒代蛋白,它们都与大肠杆菌中同源蛋白的结构相似。这些结构首先展示了在人源IPP异构酶的N端区域存在着一个不稳定的α螺旋,在空间群C2和P1的晶体结构中这个α螺旋是形成的,但是在空间群P212121的晶体结构中却打开成了一个无规则卷曲,这个α螺旋覆盖在IPP异构酶活性口袋的上方,可能控制底物的进出。然后在空间群P1的晶体结构中,发现在活性口袋中结合了一个可能来自大肠杆菌的底物类似物焦磷酸乙醇胺（EIPP）,根据这个结构我们认为IPP异构酶催化的底物IPP质子化与去质子化反应中质子的供体可能是结合在活性口袋中的一个水分子,并且很可能IPP和DMAPP采用不同的构象结合IPP异构酶。另外这三个不同晶型的晶体结构中活性相关的氨基酸的构象并不完全相同,据此我们认为人源IPP异构酶中存在着一个底物诱导的活性位点构象变化的过程。最后根据空间群P212121的晶体结构,我们提出了一个高浓度的Mn²⁺抑制人源IPP异构酶活性的机理。2.人源真核翻译起始因子eIF2B的结构生物学研究真核翻译起始过程是一个复杂的过程,其主要目的是为了使mRNA能够结合在核糖体上并使得起始Met-tRNA_i与mRNA上的AUG起始密码子正确配对,从而可以开始肽链的延伸过程。这个翻译起始过程需要很多蛋白质的帮助,它们被称为真核起始因子（eukaryotic initiation factor,eIF）。其中eIF2是携带起始Met-tRNA_i结合到核糖体的重要因子,它只有在结合GTP时才能携带起始Met-tRNA_i,但是正常情况下eIF2对GDP的亲和力要高于GTP,所以这时就需要一个鸟苷酸交换因子eIF2B的帮助把eIF2·GDP变为eIF2·GTP,这个鸟苷酸交换反应是整个翻译起始过程最重要的调控点之一。eIF2B是一个包含五个亚基的大复合物,它又可以分为调节亚复合物和催化亚复合物,其中由α,β和δ亚基组成的调节亚复合物主要应对eIF2磷酸化对eIF2B活性的抑制作用,而由γ和ε亚基组成的催化亚复合物则主要发挥催化鸟苷酸交换的活性。我们表达纯化了人源eIF2Bα亚基并用分子置换的方法解析了其2.6（?）分辨率的晶体结构,eIF2Bα的结构包含两个结构域,N端结构域和C端结构域,根据以前生化研究的结果我们描述了eIF2Bα上一个可能与eIF2或磷酸化eIF2相互作用的表面,并认为其C端结构域中的Rossmann折叠花样可能介导了其与β及δ亚基形成调节亚复合物,除此之外,我们还根据eIF2Bα的结构提出了其中两个被鉴定与一类人遗传疾病VWM相关的突变致病的可能机理。另外,我们克隆并表达纯化了人源eIF2Bε亚基的C端结构域并收集了两套不同分辨率的晶体衍射数据,这个结构域被证明是整个eIF2B复合物中最核心的发挥催化鸟苷酸交换活性的区域,其晶体结构的解析还在进行中,同时我们还克隆表达并纯化了融合MBP的人源eIF2Bβ和γ亚基蛋白。所有的这些工作为以后进一步eIF2B复合物的结构生物学研究打下了基础。更多还原

【Abstract】 1. Structural studies on human IPP isomeraseHuman IPP isomerase is an essential enzyme in isoprenoid biosynthetic pathway. It catalyzes isomerization of IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis of numerous isoprenoid products. Here we solved the crystal structure of human IPP isomerase at 1.6A resolution in space group P212121 by single-wavelength anomalous diffraction （SAD） method. High similarity between structures of human and E.coli IPP isomreases was discovered. Then we got another two different structures which belong to space group C2 and P1. The protein used for crystals belonging to space group P1 was expressed in inorganic M9 medium containing selenomethionine. These structures of human IPP isomerase show a flexible N-terminalα-helix covering the active pocket blocking the substrate entrance. Thisα-helix is formed in the structures of space group C2 and P1, but absent in the structure of space group P212121. In the structure of space group P1, a natural substrate analog ethanol amine pyrophosphate （EIPP） which is probably from E.coli was discovered binding in the active pocket. Based on this structure, a water molecule in the active pocket was proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme were also proposed. Besides, the conformations of the catalytically essential residues are not the same in these three structures and we proposed a possible substrate-induced conformation change of the active pocket for human IPP isomerase. The inhibition mechanism of high Mn²⁺ concentrations for human IPP isomerase is also discussed based on the structure of space group P212121.2. Structural studies on human eukaryotic initiation factor 2B （eIF2B）Eukaryotic translation initiation is a sophisticated process that is to ensure the binding of mRNA to ribosome and the initiator Met-tRNA_i is correctly positioned at the AUG start cordon of each mRNA. Multiple proteins named eukaryotic initiation factors （eIFs） are needed in this process. Among these, eIF2 could deliver the initiator Met-tRNA_i to the ribosome with GTP. Because that eIF2 has a higher affinity for GDP than GTP, a guanine nucleotide exchange factor eIF2B is needed to promote the exchange of GTP to GDP and results in the formation of active eIF2·GTP. complex. This reaction is one of the most important regulation points during the whole translation initiation process. eIF2B is a big complex containing five subunits. It could be divided into two subcomplexes, a regulatory subcomplex and a catalytic subcomplex. The regulatory subcomplex containing subunitsα,βand 5 sensitises eIF2B to inhibition by phosphorylation of eIF2, and the catalytic subcomplex containing subunits y and s possesses the guanine nucleotide exchange activity. We expressed and purified human eIF2Ba subunit and solved its structure at 2.6A resolution by MR method. The structure of eIF2Ba contains two domains, an N terminal domain and a C terminal domain. According to former biochemical studies, we proposed a potential binding surface on eIF2Ba for the interaction with eIF2 or phosphorylated eIF2. We also proposed that the Rossmann fold in its C terminal domain may mediate the interaction betweenα,βandδsubunits and discussed two residues in eIF2Ba of which the mutations were characterized before in a severe genetic disease VWM. Besides, we cloned, expressed, purified and crystallized a C terminal domain of human eIF2Bs subunit and collected two sets of diffraction data. The structure determination is undergoing. This segment was proven to be the core domain in eIF2B that possesses the guanine nucleotide exchange activity. In the meantime, we cloned, expressed and purified two other subunitsβandγfused with MBP tag. All these work laid solid foundation for further structural studies on the whole eIF2B complex.更多还原