STRUCTURE OF A GLYCOCONJUGATE IN SOLUTION AND IN COMPLEX WITH AN ANTIBODY FV FRAGMENT

By use of heteronuclear (C-13, H-1) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E 3G) uniformly C-13 enriched in the glucuronic acid moiety has been pro bed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple co nformations in free solution, with particularly large torsional fluctu ations about the glycosidic linkage Psi. Resonance assignments and dis tance restraints for the glycococonjugate in the bound state were obta ined from heteronuclear proton-carbon-carbon-proton-COSY and isotope-e dited NOESY techniques, respectively. Quantitation of the NOE data wit h a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond w ith 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 similar to 15 kcal/mol higher than the global minimum en ergy conformation. The glucuronide moiety undergoes a stacking interac tion with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted boun d-state conformation are in good agreement with experiment. The bound- state conformation is also in good agreement with preliminary x-ray da ta on a related complex.