The functional importance of hydrophobicity of the tyrosine at position 13 of human epidermal growth factor in receptor binding.

The tyrosine at position 13 of epidermal growth factor (EGF) has been implicated as playing a role in receptor binding due to its close proximity to the critical arginine 41 residue as well as its high degree of conservation in EGF and EGF-like proteins that can bind to the EGF receptor. Site-directed mutagenesis of tyrosine 13 in human EGF (hEGF) was employed to examine the role of this residue in ligand-receptor interaction. The removal of the hydroxyl moiety of the tyrosine by substitution with phenylalanine had little effect on the binding, indicating that it is not involved in any crucial hydrogen bonds with either the receptor or with other regions of the EGF molecule. The substitution of the aromatic tyrosine side-chain with the nonpolar leucine side-chain caused the receptor affinity to decrease only slightly, indicating that aromaticity of the amino acid at this site is also not critical. Substitutions with other hydrophobic residues, isoleucine, valine, and alanine, resulted in a significant decrease in receptor affinity as a function of decreasing hydrophobicity. Substitution of tyrosine 13 with the polar residues histidine and arginine markedly decreased receptor binding affinity, and complete removal of the side-chain by substitution with glycine dramatically lowered the binding affinity to 0.3% as compared to wild type. Analysis of three hEGF mutants, Tyr13-->Leu, Tyr13-->Arg, and Tyr13-->Gly, by circular dichroism showed that the major structural features of hEGF were not significantly altered. The results demonstrate that the decreased receptor affinities of these hEGF mutants are due to disruption of the functional contribution(s) of the tyrosine 13 residue rather than alteration(s) in the overall structural integrity. Overall, the results suggest that the tyrosine 13 side-chain plays a critical role in receptor binding by contributing to hydrophobic receptor-ligand interactions.