2-DIMENSIONAL NMR INVESTIGATIONS OF THE INTERACTIONS OF ANTIBODIES WITH PEPTIDE ANTIGENS

To increase our understanding of the molecular basis for antibody spec ificity and for the cross-reactivity of anti-peptide antibodies with n ative proteins it is important to study the three-dimensional structur e of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody c omplex but rather to concentrate on elucidating the combining site str ucture, the interactions of the antibody with its antigen and the boun d peptide conformation. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the inf ormation concerning antibody-peptide interactions and intramolecular i nteractions in the bound ligand from the crowded and unresolved spectr um of the Fab complex. These measurements yield restraints on interpro ton distances in the complex which are used to dock the peptide into c alculated models for the antibodies' combining sites. Comparison of th e interactions of three antibodies against a cholera toxin peptide (CT P3), which differ in their cross-reactivity with the toxin, yields inf ormation about the size and conformation of antigenic determinants rec ognized by antibodies, the structure of their combining sites and rela tionships between antibodies' primary structure, and their interaction s with peptide antigens.