Structure And Function Of Urm1 - Modified Protein And Escherichia Coli Protein Of Ubiquitin

Ubiquitin and Ubiquitin-like proteins (UBLs) are small modifiers that regulate many important cellular processes, via covalently conjugated to their target substrates. Eukaryotic ubiquitin-related modifier (Urm1), the most ancient UBLs, has been proven to play many important roles in yeast, such as antioxidant stress response, budding, nutrient sensing and high temperature sensitivity. It has also been regarded as a molecular fossil in the evolutionary link between eukaryotic UBLs and bacterial sulphur carriers, due to its dual role in protein and tRNA modification. Yeast tRNA-thiouridine Modification Protein1(Tum1p), a crucial component of Urml system, plays an important role in the sulphur transfer process between Nfs1p and Uba4p.In this study, Urm1,Tum1,Nfs1,Uba4fusion proteins were successfully expressed and purified in E.coli cells. Tumlp was crystallized by using the hanging-drop vapor-diffusion method at20℃. X-ray diffraction analysis revealed that the crystal belongs to tetragonal space group I4i with unit-cell parameters a=b=120.94A, c=48.35A (α=β=γ=90°), and it has a resolution of1.9A. The crystal structure of Tumlp was solved through the molecular replacement method using the2.50A resolution structure of Human3-mercaptopyruvate sulfurtransferase as a search model (Protein Data Bank entry3OLH).The structure of Tum1is comprised of11α and7β, involved in two Rhodanese-like domains(RLD). These two regions combine to form a molecule which is like an ellipsoid with a deep groove. The persulfide sulphur on the active site Cys259and an unknown ligand molecule had been found in the groove. With the support of our in vitro experiments, the unknown ligand should not be hydroxyproline.Exonuclease Ⅰ (Exol) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the3’to5’direction and has been implicated in DNA recombination and repair, such as DNA repair at an apurinic/apyrimidinic (AP) site, removement of phosphoglycolate3’-end group which introduced into DNA by oxidative damage, methyl-directed mismatch repair, frameshift mutations avoidance and etc.In this study, Exol fusion protein was successfully expressed and purified in E.coli cells. Several different types of Exol crystals were obtained, using the method of hanging-drop/sitting-drop vapor-diffusion at20℃, which are the complex of Exol with substrate-like12A-3’PHO, the complex of Exol with product-like dTTP and inactived Exol. X-ray diffraction analysis revealed that the inactived Exol crystal belongs to the space group P212121with unit-cell parameters a=67.25A, b=126.64A, c=141.25A (α=β=γ=90°), and it diffracted to at least2.52A resolution. The crystal structure of inactived Exol was solved through the molecular replacement method using the published structures of Exol as search models (Protein Data Bank entry1FXX,2QXF,3C94,3C95,3HL8,3HP9). The data showed an asymmetric unit in this crystal is assumed to contain two protein molecules, and there’s no Mg2+ion. And it was also confirmed by in vitro experiments that the chelating of Mg2+ion can totally inhibit the activity of Exol, while the polynucleotides with terminal3’-phosphoryl groups can only partly inhibit its activity. A steric hindrance hypothesis was proposed based on these facts.The structural and functional researches of Tum1and Exol were performed in this paper. We successfully solved the crystal structure of Tum1and inactived Exol and analyzed their possible mechanism.