HOW THE ANTI-(METAL CHELATE) ANTIBODY CHA255 IS SPECIFIC FOR THE METAL-ION OF ITS ANTIGEN - X-RAY STRUCTURES FOR 2 FAB' HAPTEN COMPLEXES WITH DIFFERENT METALS IN THE CHELATE

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1lambda) binds the metal-chelate hapten indium(III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding, is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those, having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-angstrom resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-angstrom resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by Nepsilon of one histidine from the heavy chain's third CDR (distance = 2.4 angstrom). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193-degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).