Structural Studies On Human Isoprenoid Pyrophosphate Isomerase And Eukaryotic Initiation Factor 2B

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.