The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor

The highly conserved asparagine residue at position 32 (Asn32)in the 'hinge' region of epidermal growth factor (EGF) separatesthe N- and C-terminal structural motifs of the EGF moleculeand is therefore an appropriate target for structure-functionstudies. Analogs of human EGF (hEGF) were generated in whichAsn32 was substituted with aspartate, glycine, isoleucine, lysine,proUne and tryptophan. The relative affinity of the EGF receptorfor mutant hEGF analogs was determined by radioreceptor competitionassay. A wide range of receptor affinities was observed dependingon the amino acid substitution. N32K and N32W hEGF analogs hadrelatively high receptor affinity, while the N32G and N32D analogsshowed decreased affinity, 35% and 25% respectively, relativeto wild type hEGF. However, no binding of the N32P analog wasdetected by radioreceptor competition assay. The N32P mutantdisplayed an NMR spectrum significantly different from thatof native wild type hEGF, indicating gross structural perturbation.In contrast, the N32K and N32D analogs exhibited spectra similarto that of native wild type hEGF. Genetically combining theN32D hEGF with an hEGF species having either the mutation L26Ghi the N-terminal region or L47A in the C-terminal region, generateddouble-mutant hEGF species whkh had relative affinities essentiallyequal to the product of the relative affinities of the parenthEGF mutants, indicating functionally independent changes inUgand-receptor interaction. These studies indicate the requirementfor H-bond donor functionality in the side chain of residuenumber 32 in forming a fully competent receptor-binding epitope.