Monoclonal antibodies as a paradigm for the high potency sweet- taste receptor

Abstract

A combination of biophysical measurements and knowledge-based computer-aided molecular modeling of antibody binding sites was used to predict the interactions between high affinity monoclonal antibodies and an intensely potent tri-substituted guanidinium sweetener. This approach permitted the prediction of the interactive residues involved in ligand binding and identification of molecular motifs that might be present in the biological sweet taste receptor. Competitive ligand binding radioimmunoassays, photoaffinity labeling and pH dependent binding experiments, site-directed mutagenesis studies and ligand induced fluorescence quenching experiments were used to determine the affinity, specificity and biophysics of the complexation. Computer-aided modeling using cDNA derived amino acid sequences for the heavy and light chain V regions of the antibodies was used to construct a three-dimensional model of the sweetener binding site. Based on these studies it was predicted that the following residues are involved in complexation: L:96 Tyr, H:33 Trp, H:96 Tyr, and L:32 Tyr form an aromatic box around the cyanophenyl ring, H:50 or H:35 Glu interact with the aryl-nitrogen, H:33 Trp is in contact with the ligand but does not involve a π-π stacking or charge-transfer interaction, and L:27[D] His and H:58 Arg also contact the ligand. Finally, the diphenyl rings of the ligand were predicted to point out of the binding site. X-ray diffraction studies of the Fab-ligand complex with a resolution of 2.2 A confirmed almost all of these predictions. The use of antibodies as a paradigm for the sweet taste receptor may provide insight into the molecular recognition motifs involved in sweet taste.