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

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-β-D-galactopyranosyl(1→6)-4-deoxy-4-fluoro-β-D-galactopyranoside (mFGal 2 ) and its selectively deuteriated analogue, methyl O-β-D-galactopyranosyl(1→6)-4-deoxy-2-deuterio-4-fluoro-β-D-galactopyranoside (me4F2dGal) 2 . The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal 2 was inferred from measurements of vicinal 1 H- 1 H coupling constants, long-range 1 H- 13 C 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 coformation of the glycosidic linkage. Analysis of 1 H- 1 H and 1 H- 13 C constants indicates that the major conformer has an extended conformation: Φ=−120 o ; ψ=180; and ω=75 o . TRNOE measurements on me4FGal 2 and me4F2dGal 2 in the presence of the specific antibody indicate that the pyranose rig 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, Φ=−152±9 o ; ψ=−128±7 o ; and ω=−158±6 o ; and a conformer with Φ=−53±- o ; Ψ=154±10 o ; and ω=−173±6 o . Neither conformation is similar to any of the observed conformations of the free disaccharide. Therefore, the X24 antibody significantly alters the conformation of its ligand upon binding. A new method, based on changes in the fluorine longitudinal relaxation rate, is used to measure the ligand-antibody dissociation rate constant. At 55 o C, this rate constant is found to be 100 s −1