PROTOONCOPROTEIN REGULATION--NMR STUDIES OF C SRC

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The proto-oncoprotein Src plays a key role in intracellular signal transduction and has been implicated in several carcinomas including breast and colon cancer. The main thrust of the proposed research is to combine structure, dynamics, and thermodynamics measurements to study the fundamental principles governing ligand binding to the SH3 and SH2 domains of this protein, and to test whether functional fragments of the Src regulatory apparatus accurately mimic function in the intact protein. The model systems employed represent Src in the "on" state, where both SH3 and SH2 domains are available to form activated signaling complexes with other proteins, some which bind to both SH3 and SH2 domains. NMR spectroscopy offers the unique ability to examine ligand binding in a system that includes protein, ligand and solvent as well as unhindered exchange between free and bound states. NMR also enables enthalpic and entropic components of binding energy to be related to structural and dynamic changes of the protein backbone and side chains upon ligand binding, and enables determination of rate constants for exchange when koff is in the fast to intermediate time scale determined by chemical shift differences between the bound and free states. Combining this information with thermodynamics measurements from isothermal titration calorimetry (ITC) allows a detailed description of enthalpic and entropic binding determinants from a unique perspective that includes the structure and dynamics of the protein in both free and bound states. While current size limitations prevent NMR studies an intact Src, protein size is not limited in ITC measurements. Hence, the validity of the widely used approach of excising functional domains for structural, dynamics, and binding studies will be tested for this system by applying NMR and ITC to the separate and covalently linked SH3 and SH2 domains, and by comparing the ITC results with those obtained for intact Src. This system offers an excellent opportunity to correlate the high resolution structure and dynamics of a protein to well defined and easily characterized functions, and to test the validity of using individual functional domains to investigate relationships between structure, dynamics and function. The proposed studies will provide valuable insights and guidance for development of novel anti-cancer agents that target the Src regulatory apparatus.