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 epidermal 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. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.
Murray
M B
MB
Protein Engineering and Molecular Mutagenesis Program and The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences 37831-8080, USA.
Tadaki
D K
DK
Campion
S R
SR
Lamerdin
J A
JA
Serpersu
E H
EH
Bradrick
T D
TD
Niyogi
S K
SK
D002942:Circular Dichroism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors