NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes

The recognition reactions between a synthetic disaccharide alpha-Kdo-(2-->4 )-alpha-Kdo-(2-->O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate liga nd. One mAb, S23-24, recognizes the disaccharides alpha-Kdo(2-->4)-alpha-Kd o-(2-->O)-allyl and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide alpha-Kdo-(2-->8)-a lpha-Kdo-(2-->O)-allyl with an approximately 10-fold higher affinity than t o the disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl. Compared to S 25-2, S23-24 binds to alpha-Kdo-(2-->4)-alpha Kdo-(2-->O)-allyl with an app roximately 50-fold increased affinity. We used NMR experiments that are bas ed on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trN OESY, and MINSY experiments, to show that the (2-->8)-specific mAb, S25-2, stabilizes a conformation of the alpha-(2-->4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of al pha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl, one that is highly populated in aq ueous 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 c arbohydrate ligand may adopt different bioactive conformations upon interac tion with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis o f bioactive conformations of carbohydrate ligands.