Two‐dimensional NMR investigations of the interactions of antibodies with peptide antigens

Abstract

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide 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. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining 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 antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with peptide antigens.

CORCEMA evaluation of the potential role of intermolecular transferred NOESY in the characterization of ligand-receptor complexes

“…This work has been motivated because of reports of intermolecular transferred NOE contacts observed experimentally (e.g., Refs. [15][16][17][18][19][20][21][22][23], as well as the realization by us [11,14] and *To whom correspondence should be addressed. A copy of the CORCEMA program may be obtained from this author.…”

“…others [10,20,21,24] that these contacts may potentially be exploited in structure refinement. Simulations of the type presented here can help clarify the complex behavior of inter-TrNOESY intensities for a wide range of conditions, and assist the experimentalist in the selection of optimal experimental conditions.…”

“…When the exchange rate is very fast on the relaxation rate scale, the intensities become independent of the exchange rates, and the analysis of the intraand inter-TrNOESY is simpler [14]. The experimental observation of inter-TrNOESY contacts between a ligand and a large macromolecule by a number of investigators [15][16][17][18][19][20][21][22][23]32] establishes that a quantitative measurement of these intensities is indeed possible for small to moderate-sized complexes. Anglister and co-workers [21] have successfully identified interTrNOESY contacts between an antibody/antigen complex using difference-NOESY methods as well as a perdeuterated antibody.…”

The principal neutralizing determinant of HIV‐1 located in V3 of gp120 forms a 12‐residue loop by internal hydrophobic interactions

“…Moreover, techniques that are used to assign resonances to the corresponding protein protons (sequential assignment) are currently not applicable for proteins of the size of the Fab fragment of the antibody [9,10]. Transferred NOE difference spectroscopy has been used to study antibody peptide complexes and to obtain simplified spectra that show exclusively crosspeaks due to antibody peptide interactions and interactions within the bound peptide [11]. Unfortunately, this technique is not applicable for the 0.5fl RP135 complex due to its very slow dissociation rate [12].…”

A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass Spectrometry Using Secondary Antibodies and Cross-Linking

“…Currently, only 60 antigen−antibody structures are in the PDB database . NMR spectroscopy has also been used but may not be suitable for the rapid determination of unknown epitopes on a protein interacting with an antibody. , …”