Secondary ligands enhance affinity at a designed metal-binding site

Background: Specific interactions of metal ions with proteins are central to all life processes. The varied functions enabled by this cooperation are a consequence of strict control of the binding-site environment, particularly the number, type and geometry of metal-coordinating sidechains. Attempts to mimic these characteristics in the de novo design of metal-binding sites have thus far concentrated primarily on metal recruitment and not on affecting site function through systematic fine-tuning of the metal environment. Results: A designed tetrahedral Zn(II)-binding site in a variant of the B1 domain of IgG-binding protein G has been expanded by introducing 'secondary ligands'. These interactions were engineered to stabilize the positions of the metal-coordinating histidine residues while retaining the desired coordination geometry. Each mutation increased the protein's affinity for metal, and combining two secondary ligands demonstrated that these enhancements are additive. These results mimic the effects of altering similar interactions observed in the native Zn(II)-binding site of carbonic anhydrase. In the B1 system, this enhanced affinity for metal is observed despite a substantial decrease in protein secondary structure. Conclusions: The intended effects of secondary ligand addition on metal affinity were observed in each mutant and demonstrated to be additive. Addition of metal also stabilized the protein's structure, partially offsetting the destabilizing effect of the mutations. These results represent a successful first attempt at designing an extended metal-binding site environment and illustrate the importance of including secondary interactions in the design of metal-binding sites.