TRANSFERRED NUCLEAR OVERHAUSER ENHANCEMENT EXPERIMENTS SHOW THAT THE MONOCLONAL-ANTIBODY STREP-9 SELECTS A LOCAL MINIMUM CONFORMATION OF A STREPTOCOCCUS GROUP-A TRISACCHARIDE-HAPTEN

Transferred nuclear Overhauser enhancement (TRNOE) experiments have be en performed to investigate the bound conformation of the trisaccharid e repeating unit of the Streptococcus Group A cell-wall polysaccharide . Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-beta-D-gl ucopyranosyl (alpha-L-rhamnopyranosyl)-alpha-L-rhamnopyranoside [C(A') B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational pref erences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous soluti on, thus displaying relatively flexible behavior. The NOE HNAc-H2A', w hich was not detected in previous work, accounts for a conformation at the beta-(1-->3) linkage with a phi angle of similar to 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was furth er complicated by spin diffusion. With the use of transferred rotating -frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that ail of the observe d long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together wit h repulsive constraints, derived from the absence of TRROE effects, we re used as input parameters in simulated annealing and molecular mecha nics calculations to determine the bound conformation of the trisaccha ride. Complexation by the antibody results in the selection of one def ined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the alpha-(1-->2) linkage when compar ed to the global minimum conformation. The results infer that the boun d conformation of the Streptococcus Group A cell-wall polysaccharide i s different from its previously proposed solution structure (Kreis et al., 1995).