Two‐Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

Abstract

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure 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 structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody-peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab-peptide complex (Fab: antibody fragment made of Fv--the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen--the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra 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 by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies' combining sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR

“…The interactions of peptides with monoclonal antibodies have been studied by NMR spectroscopy as well as X-ray diffraction. In the event of weak binding (Kd 5 1 X IO6 M -' and more importantly fast off rates, > I s", Anglister et al, 1988), methods based on the transferred nuclear Overhauser effect have enabled details of residues involved in close contact with the protein to be identified (Anglister et al, 1990(Anglister et al, , 1995, along with conformational changes in the peptides induced by binding to antibodies (Cung et al, 1991;Scherf et al, 1992). In the case of peptides that bind with high affinity, this procedure cannot be used and the complex must be studied directly.…”