Abstract
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 Iβ-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 theβ-sheet of the N-terminal subdomain. The type Iβ-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 thisβ-turn has little effect on EGF-receptor interactions.
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Abbreviations
- EGF:
-
epidermal growth factor
- h:
-
human
- m:
-
murine
- TGFα :
-
typeα transforming growth factor
- NMR:
-
nuclear magnetic resonance
- [S2A]mEGF:
-
mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine
- [S2A,S25A]mEGF and [S2A, S25P]mEGF:
-
replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively
- 125I-mEGF:
-
125I-labeled mEGF
- DMEM:
-
Dulbecco's modified Eagle's medium
- FCS:
-
fetal calf serum
- HEPES:
-
N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid
- BSA:
-
bovine serum albumin
- COSY:
-
correlated spectroscopy
- DQCOSY:
-
double-quantum filtered COSY
- NOESY:
-
nuclear Overhauser spectroscopy
- NOE:
-
nuclear Overhauser effect
- TOCSY:
-
total correlation spectroscopy
- 3 J(Hα-HN):
-
vicinal spin-spin coupling constant between amide proton and α-proton
- DSS:
-
2,2-dimethyl-2-silapentane-5-sulfonate
- δ:
-
chemical shift in ppm
- ppm:
-
parts per million,
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Lester, C.C., Wang, B., Wu, R. et al. Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26. J Protein Chem 14, 753–762 (1995). https://doi.org/10.1007/BF01886915
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DOI: https://doi.org/10.1007/BF01886915