Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody

Abstract

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR-Based, Molecular Dynamics- and Random Walk Molecular Mechanics-Supported Study of Conformational Aspects of a Carbohydrate Ligand (Galβ1-2Galβ1-R) for an Animal Galectin in the Free and in the Bound State

“…This combined NMR-and computational study of GalЈ␤1-2Gal␤1-R underscores that the different functional groups of the ligand are apparently already in a position which allows the lectin to bind this energetically favorable conformer. The present state of NMR-based analysis of galactose-containing ligand-antibody/lectincomplexes precludes a definitive conclusion on the relation of free-to bound-state ligand conformation in this specificity class of saccharide receptors (32)(33)(34)(35)(36). X-ray structures of bacterial toxins and a mammalian galectin indicate that the torsion angles at the glycosidic linkage of a bound galactose moiety in the crystal lattices are similar to those regions of low energy positions in solution (37)(38)(39)(40).…”

Conformation of surface exposed N‐terminus part of bacteriorhodopsin studied by transferred NOE technique

“…The broad signals had been cleared out by introducing an additional delay into the standard NOESY pulse sequence after the first 90 ° pulse. A non-selective 180 ° radiofrequency pulse or continious radiofrequency field have been used during this delay to prevent a distortion of the cross-peak phases [18,19]. We tested the pulse sequence MLEV-16, continious radiofrequency field as well as a train of rapidly repeated non-selective 180 ° radiofrequency pulses to attain the same goal.…”

Binding of Phenylphosphocholine−Carrier Conjugates to the Combining Site of Antibodies Maintains a Conformation of the Hapten

“…The presence of the fluorine nuclei in the TFPPC hapten provided a convenient way for measuring the dissociation rate, k off , 19 F NMR is particularly useful for studying ligand-macromolecule interactions because the 19 F nucleus is very sensitive to environmental changes (Gerig, 1989). Signals from bound ligands are often detected in 19 F spectra because there is less interference from other resonances (Craik & Higgins, 1989;Glaudemans et al, 1990) and because of typically large chemical shift dispersion. The 19 F signal was broadened and shifted upfield by 0.1 ppm when bound to the M3C65 sFv (data not shown).…”

“…Furthermore, procedures used to produce haptencarrier protein conjugates usually yield heterogeneous products, with multiple hapten units bound per carrier molecule. While this approach is advantageous for effective induction of antibody, it disallows determination of specific structural details of antigen-antibody interactions.Many structural studies of antigen-antibody interactions have focused on changes in the conformation of the hapten upon binding (Glaudemans et al, 1990;Anglister & Naider, 1991;Cheetham et al, 1991). Additional studies have emphasized structural dynamics of the antibody itself (Theriault et al, 1991; Constantine et al, 1993a,b).…”

“…Many structural studies of antigen-antibody interactions have focused on changes in the conformation of the hapten upon binding (Glaudemans et al, 1990;Anglister & Naider, 1991;Cheetham et al, 1991). Additional studies have emphasized structural dynamics of the antibody itself (Theriault et al, 1991; Constantine et al, 1993a,b).…”