METALLOANTIBODY DESIGN

Metal-binding sites were designed within the antigen-binding pocket of the catalytic antibody 43C9 based on a 3-dimensional antibody model a nd crystallographic structures of Zn-binding metalloenzymes. These tet rahedral Zn-binding sites were designed to mimic both secondary and te rtiary structural characteristics of catalytic metal sites in enzymes. Each site was planned to have two His ligands across from each other on adjacent antiparallel beta-strands. Sites were selected to sequeste r the metal ion from bulk solvent and place an open metal coordination position next to the antigen or potential substrates. Three distinct metal-site designs, with ligands in the variable light domain, in the variable heavy domain, and in both domains, were later implemented exp erimentally and shown spectroscopically to bind metal ions as predicte d. These results demonstrate the success of our design approach, the v ersatility of the antibody structure for metalloprotein design, and th e validity of the 3-dimensional model. The ability to predictably desi gn multiple metal sites in the ordered antigen-recognition region at t he bottom of the pocket allows tuning of metal ion placement and enhan ces the likelihood of interaction with putative substrates.