Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment.

By use of heteronuclear (13C, 1H) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E3G) uniformly 13C enriched in the glucuronic acid moiety has been probed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple conformations in free solution, with particularly large torsional fluctuations about the glycosidic linkage psi. Resonance assignments and distance restraints for the glycoconjugate in the bound state were obtained from heteronuclear proton-carbon-carbon-proton-COSY and isotope-edited NOESY techniques, respectively. Quantitation of the NOE data with a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond with either of the two lowest energy conformations of the free glycan, and the internal energy of the glycan in the bound state is estimated to be at most approximately 15 kcal/mol higher than the global minimum energy conformation. The glucuronide moiety undergoes a stacking interaction with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted bound-state conformation are in good agreement with experiment. The bound-state conformation is also in good agreement with preliminary x-ray data on a related complex.