2-DIMENSIONAL NMR-STUDIES OF THE INTERACTIONS BETWEEN A PEPTIDE OF CHOLERA-TOXIN AND MONOCLONAL-ANTIBODIES

To increase our understanding of the molecular basis for antibody spec ificity and for the cross-reactivity of antipeptide antibodies with na tive proteins, it is important to study the thr ee-dimensional structu re of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site st ructure, the interactions of the antibody with its antigen, and the bo und peptide conformation. To extract the information about antibody-pe ptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab-peptide compl ex (Fab: antibody fragment made of Fv-the antibody fragment composed o f the variable regions of the light and heavy chains forming a single combining site for the antigen-the light chain, and the first heavy ch ain constant regions), an nmr methodology based on measurements of two -dimensional transferred nuclear Overhauser effect (NOE) difference sp ectra was developed. Using this methodology, the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved b y specific deuteration of aromatic aromatic amino acids and specific c hain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translate d to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies' combin ing sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. (C) 1995 John Wiley & Sons, Inc.