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 human epiderm al growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N -terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Ind eed, Tyr13 has previously been shown to contribute a hydrophobic determinan t to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigate d by structure-function analysis of hEGF mutant analogues containing indivi dual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activitie s similar to wild-type indicating that an aromatic residue is not essential . Variants with polar, charged or aliphatic substitutions altered in size a nd/or hydrophobicity exhibited reduced binding and agonist activities, 1-Di mensional H-1 NMR spectra of high, moderate and low-affinity analogues at p osition 10 suggested only minor alterations in hEGF native structure. In co ntrast, a variety of replacements were tolerated at position 22 or 29 indic ating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is req uired for receptor binding, CD spectra of mutant analogues at position 22 o r 29 indicated a correlation between loss of receptor affinity and alterati ons in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas T yr22 and Tyr29 do not appear to be directly involved in receptor interactio ns. The latter conclusion, together with previous studies, suggests that hy drophobic residues on only one face of the N-terminal P-sheet of hEGF are i mportant in receptor recognition.