title: Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.
abstract: We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Ippolito
authors.forename: T T
authors.initials: TT
authors.lastname: Baird
authors.forename: S A
authors.initials: SA
authors.lastname: McGee
authors.forename: D W
authors.initials: DW
authors.lastname: Christianson
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015195:Drug Design; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D015032:Zinc / Q000378:metabolism*
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; D015032:Zinc