STRUCTURE AND SPECIFICITY OF THE ANTIDIGOXIN ANTIBODY-40-50

We determined the sequence, specificity for structurally related carde nolides, and three-dimensional. structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodi es. Measurement of the binding constants of structurally distinct digo xin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex cry stallizes in space group C2 with cell dimensions of a = 93.7 Angstrom, b = 84.8 Angstrom, c = 70.1 Angstrom, beta = 128.0 degrees. The struc ture of the complex was determined by X-ray crystallography and refine d at a resolution of 2.7 Angstrom. The hapten is bound in a pocket ext ending as a groove from the center of the combining site across the li ght chain variable domain, with five of the six complementarity-determ ining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; tw o hydrogen bonds are formed between the antibody and hapten. The surfa ce area of the antibody combining site buried by ouabain is contribute d equally by the Light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chain s. The surface complementarity between hapten and antibody is sufficie nt to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex all ows qualitative explanation of the observed fine specificities of 40-5 0, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab com plexed with digoxin, demonstrates that the antibodies bind these struc turally related haptens in different orientations, consistent with the ir different fine specificities. These results demonstrate that the im mune system can generate antibodies that provide diverse structural so lutions to the binding of even small molecules.