Structural Influence of Hydrophobic Core Residues on Metal Binding and Specificity in Carbonic Anhydrase II^,

Abstract

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…250 Alternatively, one can simply dialyze away the Zn II by soaking CA in dipicolinic acid for 10-12 days. 184,251 Metal derivatives are prepared by incubating the apoenzyme with a buffered solution of the chloride or sulfate salt of the desired metal for 24-48 h. 187,188,311 The formation of cobalt complexes can be quantified by measuring esterase activity; the formation of other metal complexes (e.g., Cu II ) may be quantified by performing a colorimetric 4-(2-pyridylazo)resorcinol assay. 188,[311][312][313] 314 The catalytically active metal ions, Zn II and Co II , coordinate to histidine residues in the active site of HCA II with distorted tetrahedral geometries, regardless of pH.…”

Carbonic Anhydrase: New Insights for an Ancient Enzyme

“…Recent studies have focused on structural features of the active site and the thermodynamics of solute association that influence metal binding specificity and avidity. The results indicate a role for hydrophobic core residues in human CA-II that are important for preorienting the histidine ligands in a geometry that favors zinc binding and destabilizes geometries that favor other metals (23,24). Calorimetric studies of CA-II and variants indicate that both desolvation of the metal ion and the binding site have major contributions to the overall thermodynamics, thus directing specificity of binding by optimizing desolvation (25,26).…”

First- and second-shell metal binding residues in human proteins are disproportionately associated with disease-related SNPs

“…Both first-and second-shell residues affect metal ion binding. Second-shell contacts contribute to binding stability by optimizing binding geometry, and to metal selectivity by discriminating between metal cations of different ionic radii and coordination geometries [Cox et al, 2000;Dudev et al, 2003]. In the tumor suppressor protein p53 (TP53; MIM# 191170), which is variant in about half of all human cancers, a zinc ion is coordinated by residues C176, H179, C238, and C242 (Protein Data Bank [PDB] identification codes [ID] 2ocj).…”