Structure-function studies of mEGF: probing the type I beta-turn between residues 25 and 26.
The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I beta-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the beta-sheet of the N-terminal subdomain. The type I beta-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this beta-turn has little effect on EGF-receptor interactions.
Lester
C C
CC
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Wang
B
B
Wu
R
R
Scheraga
H A
HA
D000818:Animals; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors