title: Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding.
abstract: Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species. The results indicate that hydrophobic residues on the exposed surface of the beta-sheet structure of the amino-terminal domain of hEGF have an important role in the formation of the active EGF-receptor complex. The substitution of hydrophobic amino acid residues, Val-19----Gly, Met-21----Thr, Ile-23----Thr, and Leu-26----Gly, resulted in decreased binding affinity, with the most severe reductions observed with the last two mutants. The mutations Ala-25----Val and Lys-28----Arg introduced amino acid residues resulting in slightly increased receptor binding affinity. Similar to previous results with acidic residues in this region [Engler, D.A., Matsunami, R.K., Campion, S.R., Stringer, C.D., Stevens, A., & Niyogi, S.K. (1988) J. Biol. Chem. 263, 12384-12390], removal of the positive charge in the Lys-28----Leu substitution had almost no effect on binding affinity, indicating the lack of any absolute requirement for ionic interactions at this site. Substitution of Tyr-22, which resulted in decreased receptor binding affinity, provides further indication of the importance of aromatic residues in this region of the molecule, as found earlier with Tyr-29 (cf. reference above).(ABSTRACT TRUNCATED AT 250 WORDS)
authors: University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Ridge 37831-8077.
authors: S R
authors: SR
authors: Campion
authors: R K
authors: RK
authors: Matsunami
authors: D A
authors: DA
authors: Engler
authors: S K
authors: SK
authors: Niyogi
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases