Cumulative effect of double-site mutations of human epidermal growth factor on receptor binding.
Using site-directed mutagenesis, it was previously found that mutation of the individual residues Tyr13, Tyr22, Ile23, or Leu26 in the amino-terminal domain or of the highly conserved Leu47 in the carboxyl-terminal domain of human epidermal growth factor (hEGF), resulted in significantly decreased receptor binding affinity. In the present study, the single-site hEGF mutants Tyr13-->His, Tyr22-->Asp, Ile23-->Thr, and Leu26-->Gly were genetically combined with the Leu47-->Ala hEGF mutant to produce a series of double-site mutant hEGF gene products having alterations simultaneously at two sites, in separate domains, within the same hEGF molecule. Similarly, the combination of the single-site hEGF mutants Tyr13-->His and Ile23-->Thr generated a double-site mutant having two mutations within the same domain. Finally, combination of the hEGF mutation Ile23-->Ala with Leu23-->Ala altered two side chains located in close proximity within the large beta-sheet region of the molecule. Analysis of the relative receptor binding affinities, determined by radioreceptor competition assays of the various single- and double-site hEGF mutants, demonstrated that mutation at any one site does not substantially alter the effect of mutation at the second site in the molecule. The cumulative effect of simultaneous mutations on relative receptor binding affinity confirms the importance of residues, including those in the large amino-terminal beta-sheet, in receptor binding, and indicates that each of the separate sites functions essentially independently in the interaction of the hEGF molecule with its receptor.
Protein Engineering and Molecular Mutagenesis Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
S R
SR
Campion
M K
MK
Geck
S K
SK
Niyogi
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004355:Drug Stability; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017978:Receptors, Growth Factor; D013329:Structure-Activity Relationship
D011956:Receptors, Cell Surface; D017978:Receptors, Growth Factor; C060000:erythroid differentiation factor receptor; D004815:Epidermal Growth Factor