Structural Zinc Site Research Articles

Self-Adaptive Dirhodium Complexes in a Metal-Organic Framework for Synthesis of N-H Aziridines.

Conformational dynamics of active sites in enzymes enable great control over the catalytic process. Herein, we constructed a metal-organic framework with conformationally dynamic active sites (Rh2-ZIF-8). The active sites in Rh2-ZIF-8 were composed of the imidazolate-bridged bimetallic center with a catalytic dirhodium moiety and structural zinc site. Even though the coordination sphere of the dirhodium species was saturated with two circularly arranged esp groups and two axial 2-MeIm ligands, it could still effectively catalyze the direct synthesis of N-H aziridines from olefins with high activity. We found that such a self-adaptive catalytic process was based on the dynamic breakage and reformation of the rhodium-zinc imidazolate bridges. Interestingly, the in situ generated dirhodium site with a unique Rh2(esp)2(2-MeIm)1 configuration was able to exhibit obviously enhanced selectivity compared to homogeneous catalyst Rh2(esp)2. Furthermore, the surrounding zinc imidazolate groups could effectively protect the dirhodium moieties from harsh environments, and this ultimately endowed it with high stability.

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy.

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

Zinc Modulates Self-Assembly of Bacillus thermocatenulatus Lipase.

Thermoalkalophilic lipases are prone to aggregation from their dimer interface to which structural zinc is very closely located. Structural zinc sites have been shown to induce protein aggregation, but the interaction between zinc and aggregation tendency in thermoalkalophilic lipases remains elusive. Here we delineate the interplay between zinc and aggregation of the lipase from Bacillus thermocatenulatus (BTL2), which is taken to be a representative of thermoalkalophilic lipase. Results showed that zinc removal disrupted the BTL2 dimer, leading to monomer formation and reduced thermostability manifesting as a link between zinc and dimerization that leads to thermostability, while zinc addition induced aggregation. Biochemical and kinetic characterizations of zinc-induced aggregates showed that the aggregates obtained from the early and late stages of aggregation had differential characteristics. In the early stages, the aggregates were soluble and possessed native-like structures, while in the late stages, the aggregates became insoluble and showed fibrillar characteristics with binding affinities for Congo red and thioflavin T. The impact of temperature on zinc-induced aggregation was further investigated, and it was found that the native-like early aggregates could completely dissociate into functional lipase forms at high temperatures while dissociation of the late aggregates was limited. To this end, we report that the zinc-induced aggregation of BTL2 can be reversed by temperature switches and initiated by ordered aggregates in the early stages that gain fibrillar-like features over time. Insights revealed by this work contributes to the knowledge of aggregation mechanisms that exist in thermophilic proteins, reflecting the potential use of metal addition and/or removal to fine-tune aggregation tendency.

Dynamic HypA zinc site is essential for acid viability and proper urease maturation in Helicobacter pylori.

Helicobacter pylori requires urease activity in order to survive in the acid environment of the human stomach. Urease is regulated in part by nickelation, a process that requires the HypA protein, which is a putative nickel metallochaperone that is generally associated with hydrogenase maturation. However, in H. pylori, HypA plays a dual role. In addition to an N-terminal nickel binding site, HypA proteins also contain a structural zinc site that is coordinated by two rigorously conserved CXXC sequences, which in H. pylori are flanked by His residues. These structural Zn sites are known to be dynamic, converting from Zn(Cys)4 centers at pH 7.2 to Zn(Cys)2(His)2 centers at pH 6.3 in the presence of Ni(ii) ions. In this study, mutant strains of H. pylori that express zinc site variants of the HypA protein are used to show that the structural changes in the zinc site are important for the acid viability of the bacterium, and that a reduction in acid viability in these variants can be traced in large measure to deficient urease activity. This in turn leads to a model that connects the Zn(Cys)4 coordination to urease maturation.

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.

The dynamics of zinc sites in proteins: electronic basis for coordination sphere expansion at structural sites.

The functional role assumed by zinc in proteins is closely tied to the variable dynamics around its coordination sphere arising by virtue of its flexibility in bonding. Modern experimental and computational methods allow the detection and study of previously unknown features of bonding between zinc and its ligands in protein environment. These discoveries are occurring just in time as novel biological functions of zinc, which involve rather unconventional coordination trends, are emerging. In this sense coordination sphere expansion of structural zinc sites, as observed in our previous experiments, is a novel phenomenon. Here we explore the electronic and structural requirements by simulating this phenomenon in structural zinc sites using DFT computations. For this purpose, we have chosen MPW1PW91 and a mixed basis set combination as the DFT method through benchmarking, because it accurately reproduces structural parameters of experimentally characterized zinc compounds. Using appropriate models, we show that the greater ionic character of zinc coordination would allow for coordination sphere expansion if the steric and electrostatic repulsions of the ligands are attenuated properly. Importantly, through the study of electronic and structural aspects of the models used, we arrive at a comprehensive bonding model, explaining the factors that influence coordination of zinc in proteins. The proposed model along with the existing knowledge would enhance our ability to predict zinc binding sites in proteins, which is today of growing importance given the predicted enormity of the zinc proteome.

Structural investigations of the nickel-induced inhibition of truncated constructs of the JMJD2 family of histone demethylases using X-ray absorption spectroscopy.

Occupational and/or environmental exposure to nickel has been implicated in various types of cancer, and in vitro exposure to nickel compounds results in the accumulation of Ni(II) ions in cells. One group of major targets of Ni(II) ions inside the cell consists of Fe(II)- and αKG-dependent dioxygenases. Using JMJD2A and JMJD2C as examples, we show that the JMJD2 family of histone demethylases, which are products of putative oncogenes as well as Fe(II)- and αKG-dependent dioxygenases, are highly sensitive to inhibition by Ni(II) ions. In this work, X-ray absorption spectroscopy (XAS) has been used to investigate the Fe(II) active site of truncated JMJD2A and JMJD2C (1-350 amino acids) in the presence and absence of αKG and/or substrate to obtain mechanistic details of the early steps in catalysis that precede O2 binding in histone demethylation by the JMJD2 family of histone demethylases. Zinc K-edge XAS has been performed on the resting JMJD2A (with iron in the active site) to confirm the presence of the expected structural zinc site. XAS of the Ni(II)-substituted enzymes has also been performed to investigate the inhibition of these enzymes by Ni(II) ions. Our XAS results indicate that the five-coordinate Fe(II) center in the resting enzyme is retained in the binary and ternary complexes. In contrast, the Ni(II) center is six-coordinate in the resting enzyme and binary and ternary complexes. XAS results indicate that both Fe(II) and Ni(II) bind αKG in the binary and ternary complexes. The electron density buildup that is observed at the Fe(II) center in the presence of αKG and substrate is not observed at the Ni(II) center. Thus, both electronic and steric factors are responsible for Ni-induced inhibition of the JMJD2 family of histone demethylases. Ni-induced inhibition of these enzymes may explain the alteration of the epigenetic mechanism of gene expression that is responsible for Ni-induced carcinogenesis.

Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing

Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.

Cadmium coordination to the zinc binding domains of the non-classical zinc finger protein Tristetraprolin affects RNA binding selectivity

Tristetraprolin [(TTP), also known as NUP475 and TIS11] is a non-classical zinc finger protein that regulates inflammatory response via a protein-RNA interaction. Specifically, TTP recognizes AU-rich RNA sequences located on the 3′ untranslated region of messenger RNA associated with cytokines. Recently, TTP was shown to be upregulated when cells were exposed to cadmium. Other types of zinc finger proteins have been shown to bind Cd(II), thus the Cd(II) binding properties of TTP were pursued. Metal binding titrations using Co(II) as a spectroscopic probe for Cd(II) were performed. Cd(II) was found to coordinate to the two Cys3His structural zinc sites of TTP (TTP-2D) with an upper-limit dissociation constant, Kd, of 3.5±0.1nM. Upon reconstitution of TTP-2D with Cd(II), the protein recognized target RNA, UUUAUUUAUUU, with a dissociation constant, Kd, of 2.4±0.2nM. The Cd(II)TTP-2D/RNA binding event was more sensitive to base mutations than the Zn(II)TTP-2D/RNA binding event. A single base mutation within the UUUAUUUAUUU oligomer decreased the Cd(II)TTP-2D binding affinity 50-fold and a double mutation decreased the affinity 1000–2000 fold. In comparison, only 2-fold and 15–25 fold changes for Zn(II)TTP-2D binding to the identical RNA sequences are measured.

Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site

The binding of zinc (Zn) ions to proteins is important for many cellular events. The theoretical and computational description of this binding (as well as that of other transition metals) is a challenging task. In this paper the binding of the Zn ion to four cysteine residues in the structural site of horse liver alcohol dehydrogenase (HLADH) is studied using a synthetic peptide mimic of this site. The study includes experimental measurements of binding constants, classical free energy calculations from molecular dynamics (MD) simulations and quantum mechanical (QM) electron structure calculations. The classical MD results account for interactions at the molecular level and reproduce the absolute binding energy and the hydration free energy of the Zn ion with an accuracy of about 10%. This is insufficient to obtain correct free energy differences. QM correction terms were calculated from density functional theory (DFT) on small clusters of atoms to include electronic polarisation of the closest waters and covalent contributions to the Zn-S coordination bond. This results in reasonably good agreement with the experimentally measured binding constants and Zn ion hydration free energies in agreement with published experimental values. The study also includes the replacement of one cysteine residue to an alanine. Simulations as well as experiments showed only a small effect of this upon the binding free energy. A detailed analysis indicate that the sulfur is replaced by three water molecules, thereby changing the coordination number of Zn from four (as in the original peptide) to six (as in water).

Medium- and short-chain dehydrogenase/reductase gene and protein families : The role of zinc for alcohol dehydrogenase structure and function.

Zinc plays an important role in the structure and function of many enzymes, including alcohol dehydrogenases (ADHs) of the MDR type (mediumchain dehydrogenases/reductases). Active site zinc participates in catalytic events, and structural site zinc maintains structural stability. MDR-types of ADHs have both of these zinc sites but with some variation in ligands and spacing. The catalytic zinc sites involve three residues with different spacings from two separate protein segments, while the structural zinc sites involve four residues and cover a local segment of the protein chain (Cys97-Cys111 in horse liver class I ADH). This review summarizes properties of both ADH zinc sites, and relates them to zinc sites of proteins in general. In addition, it highlights a separate study of zinc binding peptide variants of the horse liver ADH structural zinc site. The results show that zinc coordination of the free peptide differs markedly from that of the enzyme (one His / three Cys versus four Cys), suggesting that the protein zinc site is in an energetically strained conformation relative to that of the peptide. This finding is a characteristic of an entatic state, implying a functional nature for this zinc site.

Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: implications for an entatic state.

Zinc binding to the peptide replica and analogs to residues 93-115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the epsilon-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site.

Ferric uptake regulator protein: Binding free energy calculations and per‐residue free energy decomposition

Iron homeostasis is, in many bacterial species, mediated by the ferric uptake regulator (Fur). A regulatory site able to bind iron to activate Fur for DNA binding has been described, and a structural zinc site essential for the dimerization has also been proposed. They have been localized and named site 1 and site 2, respectively, from the crystal structure of a zinc-substituted Pseudomonas aeruginosa Fur (PA-Fur). Notwithstanding the studies on Fur proteins from various species, both the precise site of iron binding and the effect on DNA binding affinity are still controversial. These issues were investigated here by molecular dynamics simulations and free energy calculations. Simulations were performed for eight molecular systems represented by the three forms of Fur, that is, apo Fur, metal-substituted Fur, and Fur complexed with DNA. Because of the lack of a Fur-DNA complex crystal structure, the recently published model based on mass spectrometry experiments on Escherichia coli Fur (EC-Fur), and the crystal structure of PA-Fur, was used, after adjustment to adopt a symmetric conformation. The simulation results suggest that the formerly proposed site 2 is, in fact, the regulatory iron-sensing site. The calculations also predict that Fe(2+) at site 2 is hexacoordinated having an octahedral environment with only nitrogen and oxygen atoms, which is in accordance with previous spectroscopic characterizations. Energy decomposition pinpoints H87 as an additional amino acid that defines the regulatory metal site. Finally, free energy decomposition analysis reveals a number of amino acids potentially important in dimerization and in DNA binding.

The Alkylation Mechanism of Zinc-Bound Thiolates Depends upon the Zinc Ligands

Alkylation of zinc-bound thiolates occurs in both catalytic and structural zinc sites of enzymes. Recent biomimetic studies have led to a controversy as to which mechanism is operative in thiolate alkylation. Building on one of these biomimetic complexes, we have devised a series of models that allow for an appraisal of the roles of charge, ligand nature, and hydrogen bonding to sulfur on reactivity. The reactions of these complexes with methyl iodide, leading to thioethers and zinc iodide complexes, have been examined by density functional theory calculations, in the gas phase as well as in an aqueous solution. In all cases, a S(N)2 reaction is favored over sigma-bond metathesis. Both the net electronic charge and the hydrogen bond play a significant role in the nucleophilicity of the thiolate. We find that the mechanistic diversity observed experimentally can be explained by the difference in the net charge of the complexes. A dianionic complex follows a dissociative pathway, whereas an associative one is preferred for a neutral system.

Studies of the molten globule state of ferredoxin: Structural characterization and implications on protein folding and iron–sulfur center assembly

The biological insertion of iron-sulfur clusters (Fe-S) involves the interaction of (metallo) chaperons with a partly folded target polypeptide. In this respect, the study of nonnative protein conformations in iron-sulfur proteins is relevant for the understanding of the folding process and cofactor assembly. We have investigated the formation of a molten globule state in the [3Fe4S][4Fe4S] ferredoxin from the thermophilic archaeon Acidianus ambivalens (AaFd), which also contains a structural zinc site. Biophysical studies have shown that, at acidic pH, AaFd retains structural folding and metal centers. However, upon increasing the temperature, a series of successive modifications occur within the protein structure: Fe-S disassembly, loss of tertiary contacts and dissociation of the Zn(2+) site, which is simultaneous to alterations on the secondary structure. Upon cooling, an apo-ferredoxin state is obtained, with characteristics of a molten globule: compactness identical to the native form; similar secondary structure evidenced by far-UV CD; no near-UV CD detected tertiary contacts; and an exposure of the hydrophobic surface evidenced by 1-anilino naphthalene-8-sulfonic acid (ANS) binding. In contrast to the native form, this apo ferredoxin state undergoes reversible thermal and chemical unfolding. Its conformational stability was investigated by guanidinium chloride denaturation and this state is approximately 1.5 kcal mol(-1) destabilised in respect to the holo ferredoxin. The single tryptophan located nearby the Fe-S pocket probed the conformational dynamics of the molten globule state: fluorescence quenching, red edge emission shift analysis and resonance energy transfer to bound ANS evidenced a restricted mobility and confinement within a hydrophobic environment. The possible physiological relevance of molten globule states in Fe-S proteins and the hypothesis that their structural flexibility may be important to the understanding of metal center insertion are discussed.

Structural Changes of Escherichia coli Ferric Uptake Regulator during Metal-dependent Dimerization and Activation Explored by NMR and X-ray Crystallography

Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8A resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.

On the Competition for Available Zinc

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.

Sequence-Selective and Hydrolytic Cleavage of DNA by Zinc Finger Mutants

We have reported the successful conversion of the structural zinc site in zinc finger peptides to a functional zinc site. A series of resulting zinc finger mutants exhibit the hydrolytic ability of the activated ester depending on the coordination geometry and acidity of the zinc ions. In this study, we explored the hydrolytic ability of DNA by the H4 mutant since the mutant showed the highest hydrolytic ability of the activated ester among the series of mutant peptides. The zinc-bound form of the H4 mutant peptide exhibited the hydrolytic ability of activated phosphoesters and even converted the supercoiled plasmid to the nicked circular form. An increasing ionic strength leads to a loss in the nuclease ability of the zinc finger mutants due to the nonspecific interaction between the zinc finger peptide and DNA. In sharp contrast, the three-tandem H4-type zinc finger protein performed the specific DNA hydrolysis at the GC box even at a high ionic strength. Thus, the present study demonstrated that converting the native zinc site to the hydrolytic zinc site in the zinc finger protein is a novel approach for creating artificial nucleases with sequence selectivity.

Hydrolytic reaction by zinc finger mutant peptides: successful redesign of structural zinc sites into catalytic zinc sites.

To redesign a metal site originally required for the stabilization of a folded protein structure into a functional metal site, we constructed a series of zinc finger mutant peptides such as zf(CCHG) and zf(GCHH), in which one zinc-coordinating residue is substituted into a noncoordinating one. The mutant peptides having water bound to the zinc ion catalyzed the hydrolysis of 4-nitrophenyl acetate as well as the enantioselective hydrolysis of amino acid esters. All the zinc complexes of the mutant peptides showed hydrolytic activity, depending on their peptide sequences. In contrast, the zinc complex of the wild-type, zf(CCHH), and zinc ion alone exhibited no hydrolytic ability. These results clearly indicate that the catalytic abilities are predominantly attributed to the zinc center in the zinc complexes of the mutant peptides. Kinetic studies of the mutant peptides demonstrated that the catalytic hydrolysis is affected by the electron-donating ability of the protein ligands and the coordination environment. In addition, the pH dependence of the hydrolysis strongly suggests that the zinc-coordinated hydroxide ion participates the catalytic reaction. This report is the first successful study of catalytically active zinc finger peptides.