Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR.
In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Hommel
U
U
Department of Biochemistry, University of Oxford, U.K.
Dudgeon
T J
TJ
Fallon
A
A
Edwards
R M
RM
Campbell
I D
ID
D000818:Animals; D001120:Arginine / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D066246:ErbB Receptors / Q000378:metabolism; D014644:Genetic Variation; D000882:Haplorhini; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011869:Radioligand Assay; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics; D014709:Vero Cells
D014443:Tyrosine; D004815:Epidermal Growth Factor; D001120:Arginine; D066246:ErbB Receptors