The Solution Structure Of Protein Cms Sh3 Domain Of C-the Cbl C-terminal Peptide Interaction

This thesis focus on the cloning, expression, purification and structural and functional studies of domains of human disease related proteins. We cloned, expressed and purified the second SH3 domain (CMS_SH3_B) of human CMS. The structure of this domain was determined by NMR. Using Biacore and NMR chemical shift perturbation experiments, we studied the binding characteristics of CMS_SH3_B to the C-terminal peptides (Cbl-p) of c-Cbl. We determined the solution structure of CMS_SH3_B by NMR method and studied the interaction between CMS_SH3_B and Cbl-p using NMR chemical shift perturbation and biacore experiments. Our results for the first time reveal the structural details of the CMS_SH3_B and provide the evidence that the Proline-rich motif (residues 701-714) of the c-Cbl can bind to the second SH3 domain of CMS directly with a low binding affinity. We also proposed the binding mode of CMS_SH3_B with Cbl-p. All the information obtained will be valuable for the future functional study of CMS.Chapte I is the review of the physiological function and research of CMS. Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule. CMS was found as a direct binding protein of pl30Cas in a yeast-two hybrid system in search for p130Cas-interacting molecules. CMS is composed of noncatalytic protein-protein interaction domains. They are important components of integrated signal transduction pathways and of the cytoskeleton that organizes the structure of eukaryotic cells. These molecules selectively control the spatial and temporal assembly of multiprotein complexes that transmit intracellular signals that regulate cell proliferation, differentiation, and survival. The kind of structural characterization is the functional basis of CMS.In chapter II, the solution structure of CMS_SH3_B was determined by NMR spectroscope. CMS contains three SH3 domains in its NH₂ terminal region involved in the interaction of CMS and other proteins. Among the three SH3 domains of CMS, the identity (48.2%) between the second and the third SH3 domain is high than the one(42%) between the first and the second SH3 domain, suggesting that the second and the third SH3 domain may bind to the same or related ligands. It was reported that the interaction of CMS with c-Cbl mostly involves in the N-terminus SH3 domains of CMS, especially CMS_SH3_B, and the C-terminal Proline-Arginine motifs of c-Cbl. A piece of gene that encodes CMSSH3B was cloned and expressed in Escherichia coli (E.coli.). Using Ni-chelating chromatography, the recombinant protein could reach more than 90% purity. Here the solution structures of CMSSH3B were obtained by heteronuclear three-dimensional spectroscopy. The solution structure of...