Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR

A NMR study of the binding of the synthetic disaccharides alpha -Kdo-(2-->4 )-alpha -Kdo-(2-->O)allyl 1 (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid ) and alpha -Kdo-(2-->8)-alpha -Kdo-(2-->O)-allyl 2, representing partial s tructures of the lipopolysaccharide epitope of the intracellular bacteria C hlamydia, to corresponding monoclonal antibodies (mAbs) S23-24, S25-39, and S25-2 is presented. The conformations of 1 bound to mAbs S25-39 and of 2 b ound to mAbs S23-24 and S25-39 were analyzed by employing transfer-NOESY (t rNOESY) and QUIET-trNOESY experiments. A quantitative analysis of QUIET-trN OESY buildup curves clearly showed that S25-39 recognized a conformation of 1 that was similar to the global energy minimum of 1, and significantly de viated from the conformation of 1 bound to mAb S25-2. For disaccharide 2, o nly a qualitative analysis was possible because of severe spectral overlap. Nevertheless, the analysis showed that all mAbs most likely bound to only one conformational family of 2. Saturation transfer difference (STD) NMR ex periments were then employed to analyze the binding epitopes of the disacch aride ligands 1 and 2 when binding to mAbs S23-24, S25-39, and S25-2. It wa s found that the nonreducing pyranose unit was the major binding epitope, i rrespective of the mAb and the disaccharide that were employed. Individual differences were related to the engagement of other portions of the disacch aride ligands.