Zinc Finger Sites Research Articles

Model peptide for anti-sigma factor domain HHCC zinc fingers: high reactivity toward 1O2 leads to domain unfolding.

All organisms have to cope with the deleterious effects of reactive oxygen species. Some of them are able to mount a transcriptional response to various oxidative stresses, which involves sensor proteins capable of assessing the redox status of the cell or to detect reactive oxygen species. In this article, we describe the design, synthesis and characterization of Zn·LASD(HHCC), a model for the Zn(Cys)2(His)2 zinc finger site of ChrR, a sensor protein involved in the bacterial defence against singlet oxygen that belongs to the family of zinc-binding anti-sigma factors possessing a characteristic H/C-X24/25-H-X3-C-X2-C motif. The 46-amino acid model peptide LASD(HHCC) was synthetized by solid phase peptide synthesis and its Zn2+-binding properties were investigated using electronic absorption, circular dichroism and NMR. LASD(HHCC) forms a 1 : 1 complex with Zn2+, namely Zn·LASD(HHCC), that adopts a well-defined conformation with the Zn2+ ion capping a 3-helix core that reproduces almost perfectly the fold of the ChrR in the vicinity of its zinc site. H2O2 reacts with Zn·LASD(HHCC) to yield a disulfide with a second order rate constant of 0.030 ± 0.002 M-1 s-1. Zn·LASD(HHCC) reacts rapidly with singlet oxygen to yield sulfinates and sulfonates. A lower limit of the chemical reaction rate constant between Zn·LASD(HHCC) and 1O2 was determined to be 3.9 × 106 M-1 s-1. Therefore, the Zn(Cys)2(His)2 site of Zn·LASD(HHCC) appears to be at least 5 times more reactive toward these two oxidants than that of a classical ββα zinc finger. Consequences for the activation mechanism of ChrR are discussed.

Co(II) Coordination in Prokaryotic Zinc Finger Domains as Revealed by UV-Vis Spectroscopy.

Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis2 coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis2 coordination an intense d-d transition band, blue-shifted with respect to the Cys2His2 sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere.

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines.

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ββα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ββα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.

Efficient oxidation and destabilization of Zn(Cys)₄ zinc fingers by singlet oxygen.

Singlet oxygen ((1)O2) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)4 zinc finger with (1)O2 by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by (1)O2. The reaction of the ZnS4 core with (1)O2 is very fast and efficient with almost no physical quenching of (1)O2. A drastic (ca. five orders of magnitude) decrease of the Zn(2+) binding constant was observed upon oxidation. This suggests that the Zn(Cys)4 zinc finger proteins would release their Zn(2+) ion and unfold upon reaction with (1)O2 under cellular conditions and that zinc finger sites are likely targets for (1)O2.

Efficient Oxidation and Destabilization of Zn(Cys)4Zinc Fingers by Singlet Oxygen

Singlet oxygen (1O2) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)4 zinc finger with 1O2 by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by 1O2. The reaction of the ZnS4 core with 1O2 is very fast and efficient with almost no physical quenching of 1O2. A drastic (ca. five orders of magnitude) decrease of the Zn2+ binding constant was observed upon oxidation. This suggests that the Zn(Cys)4 zinc finger proteins would release their Zn2+ ion and unfold upon reaction with 1O2 under cellular conditions and that zinc finger sites are likely targets for 1O2.

Nucleophilic reactivity of Zinc-bound thiolates: subtle interplay between coordination set and conformational flexibility

During the past decades it has been established that zinc-bound cysteines in proteins can react with various electrophiles to perform physiological functions such as alkyl transfer reactions or oxidative stress sensing. Electrophiles targeting especially vulnerable structural zinc fingers have also been proposed as therapeutic agents against cancers or HIV. However, the nucleophilic reactivity of zinc fingers remains poorly understood. In this article, we investigate the nucleophilic reaction of zinc finger model peptides with H2O2 in order to get deeper insight into the factors governing the reactivity of zinc-bound cysteines of zinc finger sites. We use a set of nine peptides belonging to two different peptide families with (Cys)4, (Cys)3(His) and (Cys)2(His)2 coordination sets. One family is derived from the consensus peptide of classical ββα zinc fingers and the other one derived from the zinc finger site of the oxidative stress sensor Hsp33 that adopts a loosened zinc ribbon fold. The coordination properties and the structural behaviors of the new members of the latter family were carefully characterized. The rate constants of the reaction of the nine zinc finger models with H2O2 were measured at various temperatures to determine the activation parameters. In all cases, the reaction is characterized by a small enthalpy of activation, which shows that the nucleophilic reaction of zinc-bound cysteines is easy, and large unfavorable negative entropy of activation. Neutral Zn(Cys)2(His)2 cores are intrinsically less reactive than negatively charged Zn(Cys)4 and Zn(Cys)3(His). Interestingly, we observe that the more flexible zinc finger sites are the more reactive. Indeed the entropies of activation are strongly linked to the conformational behavior of the peptide in solution. This work reveals important factors that govern the reactivity of zinc-bound cysteines in these two structural classes of zinc fingers and can be used to identify reactive zinc fingers in proteins.

Coordination Properties of Zinc Finger Peptides Revisited: Ligand Competition Studies Reveal Higher Affinities for Zinc and Cobalt

Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.

Reactivity of polynuclear zinc‐thiolate sites

AbstractThe proton affinities of a series of mono‐ and polynuclear zinc‐thiolate compounds, mimicking zing finger protein and metallothioneins (MTs) sites, have been investigated by density functional theory calculations. This allows to evaluate the intrinsic nucleophilicity of synthetic and natural zinc‐bound thiolates and to compare their relative reactivities. The site specificity of the experimental thiolate alkylation for the Zn4Cys11 clusters in MTs is well reproduced, as well as the relative reactivities of ZnHis2Cys2, ZnHisCys3, and ZnCys4 zinc finger sites. Our results also show that terminal thiolates, bound to only one Lewis acid, are more reactive than bridging thiolates. Synthetic inorganic clusters and the Zn4Cys9His2 cluster found in a cyanobacterial MT are less reactive than Zn4Cys11 and Zn3Cys9 clusters in MTs. These results allow discussing the influence of the protein backbone and residues on the reactivity of these natural clusters. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Redox‐dependent formation of disulfide bonds in human replication protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)(4)-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H(2)O(2) treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA.

Differential reactivity of individual zinc ions in clusters from bacterial metallothioneins

The bacterial metallothionein SmtA binds four zinc ions with high affinity and specificity in a Zn 4S 9N 2 cluster. We have explored the reactivity of these zinc ions towards the metal-chelator EDTA. Under pseudo-first-order conditions, initial break-down of zinc-thiolate bonds is rapid, followed by several slower phases. The reaction with stoichiometric amounts of EDTA is relatively slow and has been followed by 1H NMR and mass spectrometry. Both methods reveal that partially metallated intermediates occur during the reaction. Three- and two-metal species are observed in only minor amounts, whereas the Zn 1 species is dominant during the mid stages of the reaction, before complete metal depletion occurs. These results suggest that the zinc finger site in SmtA is not only inert towards metal exchange, but also more resilient towards chelating agents. The greater inertness of this site may help to maintain the protein fold during metal depletion, and allow subsequent facile metal uptake. Conversely, it is likely that the protein fold is the major contributor to the observed persistence of Zn 1SmtA in this reaction. Mass spectrometric studies with His-to-Cys mutants of SmtA reveal that the primary site for EDTA attack is the His49-containing zinc site C, and that His40 has a major influence on the reactivity of three sites.

Site-Specific Detection of DNA Methylation Utilizing mCpG-SEER

Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.

Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships.

DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of approximately 40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.

Interactions of Azodicarbonamide (ADA) Species with the Model Zinc Finger Site: Theoretical Support of the Zinc Finger Domain Destruction in the HIV-1 Nucleocapsid Protein (NCp7) by ADA

Interactions of azodicarbonamide (ADA) species with zinc-containing sites, which mimic the zinc finger domains of the HIV-1 nucleocapsid protein (NCp7), have been investigated by quantum chemistry tools in order to provide theoretical insights into recent experimental findings. In the latter, reactions of ADA with NCp7 lead to destabilization of the zinc coordination sphere followed by HIV inhibition. In this work, energy profiles for the interactions of neutral and protonated ADA species with the Zn binding site have been calculated at the density functional theory level in the B3LYP approximation. The calculations show that the ADA molecule, protonated at the central nitrogen atom, reacts without activation barrier with the model zinc binding site, destructing the metal coordination sphere, while interactions of other ADA species do not lead to modification of the metal site.

Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase.

6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-finger site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium-dependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.