CUEDC2 Solution Structure And Its Interaction With The Ubiquitin NMR Studies

CUEDC2(CUE Domain Containing 2) is a protein with 287 amino acids and its molecular is 32kDa. It is found that CUEDC2 could interacts with progesterone receptor, then promotes progesterone-induced PR degradation. CUEDC2 has valuble sense to be a new therapeutic target in cancer therapy. CUEDC2 is considered to a CUE domain contained protein as a result of the analysis of bioinformatics. CUE domain contains 40 amino acids approximately that can interact with Ubiquitin traditionally. The structure of CUE domain is similar with UBA domain, both have threeα-helix and combine to the Ubiquitin through the conserved hydrophobic patch in C–terminal. In order to explain the founction and structure of CUEDC2, this thesis developed a set of experiments to study how the CUEDC2 interacts with Ubiquitin, we detected the interaction points. Meanwhile, we solved the initial structure of N-terminal of CUEDC2.We cloned, expressed and purified 15N and 15N/13C labeled protein CUEDC2(1-190), and identified by SDS-PAGE, Tricine-PAGE. We collected a series of three dimensional spectral and assigned the chemical shift of backbone by NMRPipe and NMRView.Through the virtro-biological binding experiment, We have set a series of tests on the interaction between Ubiquitin and the trunctions of CUEDC2, and identified by both SDS-PAGE and Western Blot. It was showed that CUEDC2(1-133) which without CUE domain have little hint to interact with Ubiquitin. However, both CUEDC2(133-190) and CUEDC2(1-190) were able to interact with Ubiquitin.In order to confirm the result of virtro-biological binding experiment, and to detect the specific interaction points of CUEDC2, we set up a series of titration experiment with NMR, as the result of 1H-15N HSQC spectrum, and the backbone assignment of CUEDC2(1-190), we found the specific interaction points.In order to solve the solution structral of N-terminal of CUEDC2, CUEDC2(1-133), we compared the 2D 1H-15N HSQC spectrum of CUEDC2(1-133) with CUEDC2(1-190). The result showed that little change appeared. It means that the structure of CUEDC2(1-133) and CUEDC2(1-190) were basicly consistence. We concluded it will make sense to solve the solution structure of the truction of CUEDC2, CUEDC2(1-133). We collected a series of 3D spectrum HNCACB,CBCACONH,HNCO,HNCA,HNCOCA,HCCH-COSY,HCCHTOCSY,HBHACONH,CCCONH,15N-NOESY-HSQC and 13C-NOESY-HSQC. We assigned both backbone and sidechain of CUEDC2(1-133) and abtained the initial structure.Finally, we studied the interaction between Ubiquitin and the trunction of CUEDC2. We obtained the protein of CUEDC2(1-190) and Ubiquitin by the way of cloning, expression and purification. We assigned most part of the backbone of CUEDC2(1-190) and detected the specific interaction points. Through the comparing of the 1H-15N HSQC spectrum between CUEDC2(1-133) and CUEDC2(1-190), we confirmed there was no difference in solution structure between CUEDC2(1-133) and full length protein. The result of the assignment of CUEDC2(1-133) on backbone and sidechain was helpful for the structual analysis.