Engineering a cysteine ligand into the zinc binding site of human carbonic anhydrase II

Kiefer, Laura L.; Krebs, Joseph F.; Paterno, Steven A.; Fierke, Carol A.

doi:10.1021/bi00089a004

Cited by 57 publications

(80 citation statements)

References 52 publications

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“…The optical absorbance spectrum of Co2+-T199H CAII is invariant from pH 7 to 9 and resembles the low pH spectrum of the Co2+-substituted wild-type enzyme (9), displaying broad ligand field absorption bands at 570 nm (E = 140 M-1.cm-1) and 535 nm (-= 136 M-1.cm-1). These data are consistent with a metalbound water in the coordination polyhedron of T199H CAII, indicating that H199 does not coordinate to zinc and that the pKa of this water molecule is -9, compared to 7 in the wild-type enzyme.…”

Section: Methodsmentioning

confidence: 84%

“…To accommodate the D199-Zn2+ interaction, the polypeptide backbone of the L198-T200 segment endures a significant conformational change comparable to the plastic structural changes observed in other CAII variants (8,9): the C0 atom of D199 shifts 0.6 A toward zinc, and the C0 atoms of L198 and T200 move by 0.6 and 0.5 A, respectively, from their wild-type positions. The XI and X2 torsion angles of D199 (620 and 800, respectively) are close to energetically favorable values (31).…”

Section: Methodsmentioning

confidence: 84%

“…Zinc was removed from T199D and T199E CAITs similarly at pH 6.2 except that DPA was increased to 100 mM for T199E CAII. Dissociation constants for zinc were determined by dialyzing apoenzyme against varying concentrations of zinc/DPA buffers (9,13). The [Zn]free was calculated (17) and the [ZnIbOund was determined using a 4-(2-pyridylazo)resorcinol colorimetric assay (18) X-Ray Crystallography.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…§1734 solely to indicate this fact. (8)(9)(10)(11)(12)(13) 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.…”

mentioning

confidence: 99%

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Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

Ippolito

Baird²,

McGee³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Methodsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

Ippolito

Baird²,

McGee³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Molecular dynamics simulations of human carbonic anhydrase II: Insight into experimental results and the role of solvation

Voth

1998

Proteins

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In this paper, the carbonic anhydrase II (CA II) enzyme active site is modeled using ab initio calculations and molecular dynamics simulations to examine a number of important issues for the enzyme function. It is found that the Zn2+ ion is dominantly tetrahedrally coordinated, which agrees with X-ray crystallographic studies. However, a transient five-fold coordination with an extra water molecule is also found. Studies of His64 conformations upon a change in the protonation states of the Zn-bound water and the His64 residue also confirm the results of an X-ray study which suggest that the His64 conformation is quite flexible. However, the degree of water solvation is found to affect this behavior. Water bridge formation between the Zn-bound water and the His64 residue was found to involve a free energy barrier of 2-3 kcal/mol and an average lifetime of several picoseconds, which supports the concept of a proton transfer mechanism through such a bridge. Mutations of various residues around the active site provide further insight into the corresponding experimental results and, in fact, suggest an important role for the solvent water molecules in the CA II catalytic mechanism.

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Zinc Proteomics

Nowakowski

Karim

Petering

2015

Encyclopedia of Inorganic and Bioinorganic Chemistry

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The formation of the Zn‐proteome involves a complex array of interactions of Zn 2+ with cellular proteins, beginning with transporters that govern the movement of Zn 2+ into and from the cytosol and organelles. Within these compartments, free Zn 2+ concentration is maintained at nanomolar to picomolar levels through extensive buffering by components of the proteome such as metallothionein. Current information favors the buffer as the source of Zn 2+ for the constitution of native Zn‐proteins; but neither equilibrium nor kinetic results demonstrate clearly how trafficking of Zn 2+ to specific sites occurs. The chemical basis of Zn 2+ ‐based signaling, illustrated with the transcription factor MTF‐1, is similarly ambiguous. Once formed, the Zn‐proteome is preserved under Zn 2+ ‐deficient conditions, but is susceptible to widespread metal exchange with Cd 2+ and reaction with other xenobiotics. Zn‐proteomic research extensively utilizes fluorescent sensors for Zn 2+ . Two of these, TSQ and Zinquin, form adducts with significant fractions of the Zn‐proteome and thereby reveal their status and reactivity under a variety of physiological and pathological conditions. The next step in the evolution of zinc proteomics will be to deconvolute collective Zn‐proteomic behavior into the reactions of individual Zn‐proteins. Methods are discussed that support this advance.

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Engineering a cysteine ligand into the zinc binding site of human carbonic anhydrase II

Cited by 57 publications

References 52 publications

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

Molecular dynamics simulations of human carbonic anhydrase II: Insight into experimental results and the role of solvation

Zinc Proteomics

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