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Evaluation of the role of electrostatic residues in human epidermal growth factor by site‐directed mutagenesis and chemical modification
Author(s) -
Niyogi Salil K.,
Campion Stephen R.,
Tadaki Douglas K.
Publication year - 1992
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240500108
Subject(s) - lysine , arginine , glutamine , alanine , glutamic acid , biochemistry , chemistry , amino acid , site directed mutagenesis , binding site , receptor , mutagenesis , epidermal growth factor , glutamate receptor , stereochemistry , biology , mutation , mutant , gene
Four residues in the carboxy‐termianl domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site‐directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor‐ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge‐conservative replacement of glutamate 40 with asparate resulted in a decrease in receptor binding affinity to 30% relative to wild‐type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitutiono of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side‐chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274–2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor‐ligand association. The most highly conserved of the four residues studied in this report, aspartate 46, was replaced with alanine, tyrosine, and arginine, resulting in a decrease in relative receptor affinity to 23, 14, and 4 percent, respectively, and suggests the importance of an acidic group at this site of EGF. The ability to generate sufficient yields of mutant recombinant EGF protein was sensitive to the type of side‐chain substitutions generated at the sites described in this report and may indicate a role for these residues in the formation of the EGF structure apparently required for productive yields of EGF proteins in the expression system used in this study. © 1992 Wiley‐Liss, Inc.