RECOGNITION OF AN ANTIPARALLEL BETA-SHEET STRUCTURE OF HUMAN EPIDERMAL GROWTH-FACTOR BY ITS RECEPTOR - SITE-DIRECTED MUTAGENESIS STUDIES OF ALA-30 AND ASN-32

The Ala-30 and Asn-32 residues involved in the major antiparallel beta-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 beta-sheet structure of hEGF. a wider contact area than previously suggested.