Structure-function analysis of a conserved aromatic cluster in the N- terminal domain of human epidermal growth factor

The importance of a cluster of conserved aromatic residues of humanepidermal growth factor (hEGF) to the receptor binding epitope is suggestedby the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 ofthe N-terminal beta-sheet to form a hydrophobic surface on the hEGFprotein. Indeed, Tyr13 has previously been shown to contribute ahydrophobic determinant to receptor binding. The roles of His10, Tyr22 andTyr29 were investigated by structure-function analysis of hEGF mutantanalogues containing individual replacements of each residue. Substitutionswith aromatic residues or a leucine at position 10 retained receptoraffinities and agonist activities similar to wild- type indicating that anaromatic residue is not essential. Variants with polar, charged oraliphatic substitutions altered in size and/or hydrophobicity exhibitedreduced binding and agonist activities. 1- Dimensional 1H NMR spectra ofhigh, moderate and low-affinity analogues at position 10 suggested onlyminor alterations in hEGF native structure. In contrast, a variety ofreplacements were tolerated at position 22 or 29 indicating that neitheraromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptorbinding. CD spectra of mutant analogues at position 22 or 29 indicated acorrelation between loss of receptor affinity and alterations in hEGFstructure. The results indicate that similar to Tyr13, His10 of hEGFcontributes hydrophobicity to the receptor binding epitope, whereas Tyr22and Tyr29 do not appear to be directly involved in receptor interactions.The latter conclusion, together with previous studies, suggests thathydrophobic residues on only one face of the N-terminal beta-sheet of hEGFare important in receptor recognition.