The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 5744), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.One of the most intriguing aspects of antibody recognition is the ability of some antibodies raised against peptide fragments of proteins to crossreact with intact native proteins (1-3). This phenomenon not only has considerable significance from the point of view of protein structure and folding but also has generated much interest in the possibility ofusing small peptides in a pharmacological role, particularly as synthetic vaccines (4,5). Little is known, however, about the underlying principles that determine whether a particular peptide is able to act as a good mimic of a protein epitope, primarily-because of the shortage of structural information pertaining to antibodies complexed with their peptide antigens. To our knowledge there has, for example, been only one report ofthe crystallographic structure of an anti-peptide antibody-peptide complex (6). In contrast a number of structures of anti-protein antibodies complexed with their protein antigens have been determined (7-11). As part of a long-term study in our laboratories of the structural origins of antibody recognition and protein-peptide crossreactivity, a panel of antibodies (Gloopl-GloopS) specific for the loop determinant of hen egg lysozyme was raised against a peptide immunogen that incorporated residues 57-84 of the protein sequence (12)-known as the "loop" peptide. This sequence corresponds to a large surface loop in the native protein structure and contains a disulfide bridge between residues 64 and 80. The NMR studies have focused on one of the antibodies, Gloopl. The antibody binds two forms of the loop peptide, where the disulfide is either reduced or oxidized, with equal affinity (Kd 10-8 M). The antibody is also strongly crossreactive with the native protein; it binds to hen lysozyme itself with only a 10-fold drop in affinity (12, 13). Crystallographic studies have yielded a structure for the Gloopl Fab (R.E.G. a...

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2 . Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80-or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/ min per mg of EGF receptor protein at 0°C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22°C.Tyrosine phosphorylation has become recognized as a protein modification reaction with potential roles in regulation of cell metabolism, reproduction, and differentiation. Viral transforming gene products and their normal cell homologues (1-4) and receptors for epidermal growth factor (EGF) (5), platelet-derived growth factor (6-8), and insulin (9-10) all catalyze transfer of phosphate from ATP to tyrosine residues of proteins. The best-characterized substrates for these kinases are the kinase proteins themselves; all undergo autophosphorylation. In addition, viral transformation of cells or binding of ligands to specific cell surface receptors stimulates tyrosine phosphorylation of cellular proteins (11-13). With the exception of vinculin (14) (20). To test for possible involvement of tyrosine phosphorylation in regulation of steroid receptor function, we examined purified hen oviduct progesterone receptor as a substrate for purified EGF receptor kinase. EXPERIMENTAL PROCEDURESPurification of Progesterone Receptor. The transformed progesterone receptor was purified from hen oviducts by a procedure similar to that of Chang et al. and B (105-kDa) subunits (kDa, kilodaltons). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/ PAGE) analysis of purified preparations showed minor impurities migrating at 280, 150, and 45 kDa on gels overloaded for receptor protein. In addition, there were faint bands immediately below the 80-and 105-kDa receptor bands that could arise from limited proteolysis; these increased when the serine protease inhibitor phenylmethylsulfonyl fluoride was omitted during receptor preparation. Receptor preparations were concentrated at the final step by precipitation with (NH4)2SO4 and dissolved in, and dialyzed against, 20 mM Hepes, pH 7.4. Photoaffinity labeling of the receptor subunits with 3H-labeled 17a, 21-dimethy...

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