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 -- nu-cleophile. 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.One goal of metalloprotein (re)design is to develop a rationale for the de novo construction of high-affinity metal ion sites in proteins that serve particular catalytic, regulatory, or structural functions. To date, modest progress has been reported in the construction of a-helical bundles (1, 2) and antibodies (3-5) that bind transition metals with micromolar affinity. Additionally, regulatory metal-binding sites have been created in trypsin (6) and glycogen phosphorylase (7) and characterized by x-ray crystallography. We currently report the design and x-ray crystallographic structure determination of a carbonic anhydrase II (CAII) variant with femtomolar zinc affinity; enhanced affinity is achieved by insertion of an additional protein ligand into the metal coordination polyhedron. This strategy was guided by previous work with threonine-199 --cysteine (T199C) CAII, in which a 4-fold enhancement of zinc affinity was achieved; here, the engineered cysteine side chain displaces zinc-bound solvent to form a H3C-Zn2+ site (8, 9).The zinc-binding site of CAII is an outstanding paradigm for structure-assisted design experiments since this metalloprotein is easily manipulated at the genetic level, is highly overexpressed in Escherichia coli, and is readily crystallized for high-resolution x-ray crystallographic structure determinationThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(8-13). The 1.54-A resolution crystal structure of CAII (14) Inspired by the zinc-binding behavior of the T199C CAII variant, modeling studies suggested that substitution of amino acids with longer side chains at position 199 would further enhance zinc binding. Therefore, we replaced T199 with the potential metal ligand residues of aspartate, glutamate, and histidine. The three-dimensional structures of threonine-199 -_ aspartate (T199D) and threonine-199 --glutamate (T199E) CAlls reveal the successful addition of a fourth protein ligand to zinc, and metal affinity is enhanced 200-fold for the T199E CAII variant. However, the structure of threonine-199 -> histidine (T199H) CAII reveals that the engineered histidine residue does not coordinate to zinc and metal affinity is compromised 20-fold. Not surprisingly, the catalytic activity of all three CAII variants is decreased by more than three orders of magnitu...

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

“…The positioning and apparent functioning of Thr 325 in TrzN has a known precedent in carbonic anhydrase (CA), a protein that is in a different structural family but was observed here to have a similar active site (32)(33)(34)(35)(36). The overall folds differ markedly.…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…The fourth ligand in ZnuA is either water [7,13] or nonconserved Glu59 as described here and elsewhere [14]. The biological significance of interchanging Glu59 and water is not clear but could significantly modulate the Zn 2+ binding affinity [48]. The third structural feature linked to specificity is the presence of the His-rich loop in the Zn 2+ binding proteins [1,2].…”

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

“…This is illustrated by protein engineering experiments with the enzyme carbonic anhydrase that binds zinc by the same tetrahedral co-ordination chemistry that is observed in the colicin endonucleases, with an equilibrium dissociation constant of 4 pM (43). The affinity for zinc increases 200-fold when a fourth protein ligand is inserted into the site normally occupied by the hydrolytic water molecule (44).…”