Selection of Carbonic Anhydrase Variants Displayed on Phage

Abstract

In all metalloenzymes, hydrophobic residues surround the metal binding site. In carbonic anhydrase II (CAII) residues Phe 93 , Phe 95 , and Trp 97 flank two of the three histidines that coordinate zinc to form a hydrophobic cluster beneath the zinc binding site. A library of CAII variants differing in these hydrophobic amino acids was prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity. Wild-type CAII was enriched 20-fold by selection, and consensus residues at each position were identified from the enriched CAII variants (Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and Trp and Val at position 97). Highly selected variants have zinc affinity and catalytic activity nearly equal to that of wild-type CAII, indicating that the aromatic residues are not absolutely essential. However, the zinc dissociation rate constant and catalytic activity of the variants correlate with the volume of the amino acids at positions 93, 95, and 97. In summary, metalloenzyme variants displayed on phage can be selected on the basis of metal affinity; such methods will be useful for optimization of metal ion biosensors.Understanding the structural determinants of metal ion affinity in proteins is an important step in the design of protein metal sites. Such sites may be used to stabilize proteins, to regulate the activity of proteins, or for use in biosensors to quantify trace metal ions (see reviews in Refs. 1-3). Because of its high affinity and specificity for zinc, the His 3 metal polyhedron of carbonic anhydrase (CAII) 1 often has been used as a model for designing metal sites in existing proteins (4 -6) and in de novo proteins such as the minibody (7) and four helical bundle protein (8, 9). While the protein metal ligands of CAII have been incorporated into these proteins with coordination geometry very similar to that observed in CAII, these designed metal sites lack the zinc avidity and catalytic activity of biological zinc sites, suggesting that further protein structural factors in CAII contribute to the metal affinity and reactivity of this enzyme.To probe the relationship between protein structure and metal ion affinity and specificity, many of the conserved structural features near the zinc binding site of CAII have been investigated (review see Ref. 10). The refined x-ray crystal structure of CAII (11) 199 (11). This conserved network of residues that form hydrogen bonds with the His ligands enhances zinc affinity (15-17); the Q92A and E117A substitutions both decrease zinc affinity 5-10-fold and increase the zinc dissociation rate constant. Therefore, the protein structure surrounding the zinc binding site of CAII is finely tuned for high zinc affinity and slow rate constants of metal dissociation.A further structural feature observed in all metalloproteins is that the hydrophilic direct metal ligands are embedded within a larger shell of hydrophobic groups (18). In CAII, residues Phe 93 , Phe 95 , and Trp 97 flank the...

Kinetics of metal binding by the toxic metal-sensing transcriptional repressor Staphylococcus aureus pI258 CadC

“…If these observed rates of CadC-metal encounter complex formation reflect true bimolecular rate constants, they are equal to, or orders of magnitude larger, than those reported for other metalloproteins, including Bacillus cereus b-lactamase (10 7 M À1 s À1 for Zn(II)) [45], Zn(II) E9 endonuclease (10 6 M À1 s À1 ) [28], Mn(II) FosA (3 · 10 5 M À1 s À1 ) [27], Bi(III)-transferrin (2 · 10 5 M À1 s À1 ) [46], Zn(II) carbonic anhydrase (10 5 M À1 s À1 ) [47] and Cu(II)-azurin (2.2 · 10 3 M À1 s À1 ) [37]. Although rapid, k obs1 for Pb(II)-CadC complex is slower than association rate constant for the binding of Zn(II) to reduced glutathione [48].…”

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

“…187 Fierke and co-workers conducted a series of studies in which they measured the affinities of different metals to HCA II and its active site mutants to determine the basis of selectivity for zinc in this protein. 251,311,321,322 The investigators looked at the influence of hydrophobic core residues Phe93, Phe95, and Trp97 on binding of metals and found that substitution of those residues with smaller side chains reduced the affinity of the protein to Zn II and Co II but increased the affinity to Cu II . 311,321 X-ray analysis of the mutants revealed new cavities formed in the core of the protein and shifts in the positions of metal-coordinating His94 and secondshell ligand Gln92.…”

“…251,311,321,322 The investigators looked at the influence of hydrophobic core residues Phe93, Phe95, and Trp97 on binding of metals and found that substitution of those residues with smaller side chains reduced the affinity of the protein to Zn II and Co II but increased the affinity to Cu II . 311,321 X-ray analysis of the mutants revealed new cavities formed in the core of the protein and shifts in the positions of metal-coordinating His94 and secondshell ligand Gln92. 251 The investigators concluded that the aromatic residues, although they do not directly coordinate the metal, preorient the metal-binding side chains in such a fashion as to favor tetrahedral zinc-binding geometry and to destabilize alternative geometries.…”

Redox Switch of Hsp33 Has a Novel Zinc-binding Motif

“…Ϫ1 at pH 7.0, 30°C) (20). Noteworthy, incubation of 40 M wild type Hsp33 in 2 mM TPEN showed that the apparent half-time of zinc release was only 160 Ϯ 10 min at 23°C.…”