STRUCTURAL PREDICTIONS OF THE BINDING-SITE ARCHITECTURE FOR MONOCLONAL-ANTIBODY NC6.8 USING COMPUTER-AIDED MOLECULAR MODELING, LIGAND-BINDING, AND SPECTROSCOPY

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p- cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affini ty (K-d = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbanc e spectroscopy, and fluorescence spectroscopy, we have predicted the s tructure of the variable domain fragment (Fv) and identified the key r esidues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and comp lexation. Most notable are H:33W, which is responsible for ligand-indu ced tryptophan fluorescence quenching, H:56R; which forms a salt bridg e with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligan d. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 Angstrom) and the Fab-ligand comple x (2.2 Angstrom). Comparisons between the theoretical model and experi mental native and liganded Fab structures are made.