The Ala-30 and Asn-32 residues involved in the major antiparallel β-sheet structure of human epidermal growth factor (hEGF) were substituted with various amino acid residues, and the receptor-binding affinities of the nine variant hEGFs were determined by the use of human KB cells. The Ala-30→Arg. Ala-30→His and Ala-30→Phe substitutions drastically reduced the binding affinity, suggesting that the side chain in position 30 of Ala-30 of hEGF is required to be small for the receptor binding. The Asn-32→Asp substitution significantly reduced the binding affinity, while the Asn-32→His variant could bind to the receptor as well as to the wild-type hEGF. Therefore, it seems to be important for receptor binding that the side chain in position 32 does not have a negative charve but does have an NH group. Thus, we propose that, in the ligand-receptor complex, the receptor recognizes, on one side of the antiparallel β-sheet structure of hEGF, a wider contact area than previously suggested.
