STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC-BINDING SITE WITH FEMTOMOLAR AFFINITY

We have inserted a fourth protein ligand into the zinc coordination po lyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (K-d = 20 fM). The three-dimensional structures of threonine -199 --> aspartate (T199D) and threonine-199 --> glutamate (T199E) CAI Is, determined by x-ray crystallographic methods to resolutions of 2.3 5 Angstrom and 2.2 Angstrom respectively, reveal a tetrahedral metal-b inding site consisting of H94, H96, H119, and the engineered carboxyla te side chain, which displaces zinc-bound hydroxide. Although the ster eochemistry of neither engineered carboxylate-zinc interaction is comp arable to that found in naturally occurring protein zinc-binding sites , protein-zinc affinity is enhanced in T199E CAII demonstrating that l igand-metal separation is a significant determinant of carboxylate-zin c affinity. In contrast, the three dimensional structure of threonine- 199 --> histidine (T199H) CAII, determined to 2.25-Angstrom resolution , indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker z inc-binding site. All three of these substitutions nearly obliterate C O2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein lig and represents a general approach for increasing protein-metal affinit y if the side chain can adopt a reasonable conformation and achieve in ner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity meta l ion biosensors.