NMR-STUDY OF THE TRANSFORMING GROWTH-FACTOR-ALPHA (TGF-ALPHA)-EPIDERMAL GROWTH-FACTOR RECEPTOR COMPLEX - VISUALIZATION OF HUMAN TGF-ALPHA BINDING DETERMINANTS THROUGH NUCLEAR OVERHAUSER ENHANCEMENT ANALYSIS

The study of human transforming growth factor-alpha (TGF-alpha) in com plex with the epidermal growth factor (EGF) receptor extracellular dom ain has been undertaken in order to generate information on the intera ctions of these molecules. Analysis of H-1 NMR transferred nuclear Ove rhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involv ed in contact with the larger protein. Significant increases and decre ases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that resi dues of the A- and C-loops of TGF-alpha form the major binding interfa ce, while the B-loop provides a structural scaffold for this site. The se results corroborate the conclusions from NMR relaxation studies (Ho yt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and S ykes, B. D. (1994) Biochemistry 33, 15283-15392), which suggest that t he C-terminal residues of the polypeptide are immobilized upon recepto r binding, while the N terminus of the molecule retains considerable f lexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These res ults give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-bin ding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.