Metalloantibody design.

Abstract

Metal-binding sites were designed within the antigen-binding pocket of the catalytic antibody 43C9 based on a 3-dimensional antibody model and crystallographic structures of Zn-binding metalloenzymes. These tetrahedral Zn-binding sites were designed to mimic both secondary and tertiary 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 sequester 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 experimentally and shown spectroscopically to bind metal ions as predicted. These results demonstrate the success of our design approach, the versatility of the antibody structure for metalloprotein design, and the validity of the 3-dimensional model. The ability to predictably design multiple metal sites in the ordered antigen-recognition region at the bottom of the pocket allows tuning of metal ion placement and enhances the likelihood of interaction with putative substrates.