Structure-based minimization of transforming growth factor-alpha (TGF-alpha) through NMR analysis of the receptor-bound ligand. Design, solution structure, and activity of TGF-alpha 8-50.
The investigation of a N-terminally truncated human transforming growth factor-alpha (TGF-alpha; residues 8-50) has been completed to determine the contribution of the N terminus to receptor binding and activation. The deletion protein was proposed and designed through study of NMR relaxation and nuclear Overhauser enhancement data obtained from the TGF-alpha-epidermal growth factor (EGF) receptor complex, which indicated that the residues N-terminal to the A loop remain flexible in receptor-bound TGF-alpha and thus suggested their lack of involvement in receptor binding (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292; McInnes, C., Hoyt, D. W., Harkins, R. N., Pagila, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1996) J. Biol. Chem. 271, 32204-32211). TGF-alpha 8-50 was shown to have approximately 10-fold lower affinity for the receptor than the native molecule in an assay quantifying the ability to compete with EGF for binding and to have a similar reduction in activity as indicated by a cell proliferation assay. NMR solution structural calculations on this molecule demonstrate correct formation of the three disulfide bonds of TGF-alpha 8-50 and have established the presence of native secondary structure in the B and C loops of the protein. However, some perturbation of the global fold with respect to the orientation of the subdomains was observed. These results suggest that although the N-terminal residues do not contribute directly to binding, they make a significant contribution in defining the conformation of the growth factor, which is required for complete binding and activity and is therefore significant in terms of producing native folding of TGF-alpha. They also show that information obtained from the receptor-bound ligand can be used to guide the design and minimization of TGF-alpha analogues. The implications of the study of TGF-alpha 8-50 for the design and synthesis of reductants of this growth factor are therefore discussed.
Protein Engineering Network of Centres of Excellence, 713 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
C
C
McInnes
J
J
Wang
A E
AE
Al Moustafa
C
C
Yansouni
M
M
O'Connor-McCourt
B D
BD
Sykes
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003198:Computer Simulation; D066246:ErbB Receptors / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011522:Protons; D012996:Solutions; D013499:Surface Properties; D016211:Transforming Growth Factor alpha / Q000737:chemistry* / Q000494:pharmacology
D008024:Ligands; D010446:Peptide Fragments; D011522:Protons; D012996:Solutions; D016211:Transforming Growth Factor alpha; C115149:transforming growth factor-alpha (8-50); D066246:ErbB Receptors