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NMR Experiments Reveal Distinct Antibody-Bound Conformations of a Synthetic Disaccharide Representing a General Structural Element of Bacterial Lipopolysaccharide Epitopes

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journal contribution
posted on 24.04.1999 by Thomas Haselhorst, Juan-Felix Espinosa, Jesus Jiménez-Barbero, Tobias Sokolowski, Paul Kosma, Helmut Brade, Lore Brade, Thomas Peters
The recognition reactions between a synthetic disaccharide α-Kdo-(2→4)-α-Kdo-(2→O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate ligand. One mAb, S23-24, recognizes the disaccharides α-Kdo-(2→4)-α-Kdo-(2→O)-allyl and α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl with an approximately 10-fold higher affinity than to the disaccharide α-Kdo-(2→4)-α-Kdo-(2→O)-allyl. Compared to S25-2, S23-24 binds to α-Kdo-(2→4)-α-Kdo-(2→O)-allyl with an approximately 50-fold increased affinity. We used NMR experiments that are based on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trNOESY, and MINSY experiments, to show that the (2→8)-specific mAb, S25-2, stabilizes a conformation of the α-(2→4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of α-Kdo-(2→4)-α-Kdo-(2→O)-allyl, one that is highly populated in aqueous solution and another conformation that is similar to the one bound by S25-2. This is the first example where it is experimentally shown that a carbohydrate ligand may adopt different bioactive conformations upon interaction with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis of bioactive conformations of carbohydrate ligands.