(EGF) by 1H NMR provides evidence that

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epidermal growth factor (EGF) by 1H NMR provides evidence that leucine-47 is involved in the interactions with the EGF receptor. (transforming growthfactor ...
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 9836-9840, December 1989 Biophysics

Conformational characterization of a single-site mutant of murine epidermal growth factor (EGF) by 1H NMR provides evidence that leucine-47 is involved in the interactions with the EGF receptor (transforming growth factor a/protein engineering/protein structure and function/rational drug design)

FRANKLIN J. MOY*, HAROLD A. SCHERAGA*, JIN-FU LIut, RAY WUt, AND GAETANO T. MONTELIONEt *Baker Laboratory of Chemistry, Cornell University, Ithaca, NY 14853-1301; tSection of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853-2703; and tCenter for Advanced Biotechnology and Medicine, and Department of Chemistry, Rutgers University, Piscataway, NJ 08854-5635

Contributed by Harold A. Scheraga, September 28, 1989

Receptor-binding studies (16) demonstrate reduced binding activities for both of these analogs in which the side chain of Leu-47 has been replaced by the shorter side chains of valine and serine, respectively. Similar observations have been made for [His47]hEGF (17) and [Ile47]hTGF-a (18). These results demonstrate that Leu-47 is important for biological activity. The Leu-47 residue is either directly involved in the interactions between these growth factors and their receptor or indirectly involved by determining the conformation of a binding site in some other part of the molecule. However, it is not possible from these mutation studies alone to separate direct effects of side-chain substitutions on ligand-receptor interactions from indirect effects resulting from perturbations of the protein structure remote from the site of mutation. To identify the spatial structural changes that form the basis for reduced binding activity, we have now determined nearly complete sequence-specific 1H NMR assignments for [Ser47]mEGF and compared these with the corresponding assignments for wt mEGF. Because these proton resonance frequencies are affected by protein structure, dynamics, and solvent accessibility, this approach samples simultaneously many conformational probes at known sites throughout the protein. The data presented in this paper demonstrate that no significant conformational changes occur in locations remote from the three-dimensional site ofthe Leu-47 to Ser mutation.

Epidernal growth factor (EGF) is a small protein containing 53 amino acids and three disufide bonds. There is significant current interest in structure-function relaABSTRACT

tionships in EGF and EGF-like proteins, including the homol-

ogous type-a transforming growth factors. The Leu47 residue of murine EGF (mEGF) is one of several that are strongly conserved among the EGF-Iike growth factors, suggesting that it may contribute to the active site of mEGF. In several different binding assays, the activity of the mutant analog in which Leu-47 is replaced by Ser ([Ser47]mEGF) ranges from 8 to 18 times weaker than that of wild-type mEGF. The NMR data summarized in this paper demonstrate that the sicant differences in the binding activities of wild-type and [Ser47]mEGF cannot be attributed to structural changes remote from the threedimensional site of mutation. The only minor conformational changes that are indicated by these data involve side chains of residues proximal to Leu-47 in the three-dimensional structure. Therefore, Leu-47 and/or residues spatially adjacent to Leu-47 constitute part of the active site of mEGF.

Epidermal growth factor (EGF) is a small protein containing 53 amino acids and three disulfide bonds. There is significant current interest in structure-function relationships in EGF and EGF-like proteins, including the homologous type a transforming growth factor (TGF-a). EGF and TGF-a bind to the same membrane-bound receptor. These growth factors and their receptor play a central role in regulating growth and differentiation of various epidermal and epithelial tissues and cell lines and are involved in the molecular basis of oncogenesis and wound healing (1-3). Although x-ray crystal structures are not yet available for any of these proteins, low-resolution three-dimensional structures for murine EGF (mEGF) (4-6), human EGF (hEGF) (7-9), rat EGF (10), and human TGF-a (hTGF-a) (11-14) in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy. These structures provide a rational basis for interpreting the effects of site-directed mutation on biological properties of these proteins and for the design of growth factor analogs. The Leu-47 residue is one of several that are strongly conserved among the EGF-like growth factors, suggesting that it may contribute to the active site of mEGF. Truncated forms of mEGF in which the C-terminal polypeptide segment containing Leu-47 is disturbed, including mEGF-(1-47) and mEGF-(1-46), exhibit diminished binding activities (15). We have cloned and expressed (16) a synthetic gene for mEGF in Escherichia coli and have generated and purified wild-type (wt)- and mutant [Val47]- and [Ser47]mEGF molecules in which Leu-47 is replaced by valine and serine, respectively.

MATERIALS AND METHODS Recombinant wt and [Ser47]mEGF molecules were expressed in E. coli and purified by methods described elsewhere (16). These preparations were found to be >98% homogeneous by C18 (Waters) reversed-phase analytical HPLC using various gradients of acetonitrile in 0.09% trifluoroacetic acid with absorbance detection at 205 nm and by anion-exchange chromatography on a Pharmacia analytical Mono Q column with absorbance detection at 280 nm. The homogeneity and identity of these molecules were also confirmed by amino acid analysis (16). Reversed-phase and anion-exchange chromatography subsequent to completion of the 1H NMR measurements show negligible (