Specificity Of YGGA2_GAE Domain For Its Accessory Proteins Assembly

There are three parts in this dissertation:Part one:Specificity of yGGA2_GAE domain for its accessory proteins assemblyVesicle transport and secretion process are the basic events of life activities. Clathrin-coated vesicles play a major role in the Golgi to the lysosome transport. In yeast, GGA2, Ent3, Ent5 are three important adapter proteins involving in this process. Ent3 and Ent5 are similar proteins;they have redundant functions, but also have many different characteristics. The GGA2-Ent3/5 interactions were suggested to be an important link to functional differentiation of Ent3/5. It was also suggested that there were a few motifs in Ent3/5 would bind to GGA2. In this work, we identified that 2 of 3 motifs in Ent3 and 1 of 2 motifs in Ent5 can bind to GGA2. And we also resolved the crystal structure of GAE domain of GGA2 (which is concerned in this interaction) at 1.8A resolution. Structural docking and mutagenesis analysis show inactive motifs in Ent3/5 repel yGGA2 GAE binding through the repulsion at position+1 and+3. We further propose a model of the GGA2-Ent3/5 synergy.Part two:structural biological research on protein L-isoaspartyl methyltransferase from Escherichia coliThe formation of isoaspartyl linkages through deamidation of asparagine and isomerization of aspartate is a common type of protein damage. The protein L-isoaspartyl methyltransferase (PIMT) is thought to recognize L-isoaspartyl residues and repair this kind of damaged proteins. Curiously, the PIMTs in bacteria have lower activity than in mammalian. We resolved the crystal structure of Escherichia coli PIMT (EcPIMT) at a resolution of 2.1 A, with its cofactors bound. Based on a structural analysis, we speculate that the strong combine power between the protein and its cofactor leads to the lower activity of EcPIMT.Part three:Crystallization and preliminary X-ray analysis of Escherichia coli RNase GAs a kind of specifical endoribonuclease, RNase G has a demonstrated role in the degradation of mRNA, processing of ribosomal RNA. Additionally, this family may also have a role as a cytoskeletal element either in cell division or chromosome segregation. We expressed and crystallized RNase G from Escherichia coli providing a foundation for further study of its structural features and biochemical functions.