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.     

Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Novel strategy for the design of a new zinc finger: creation of a zinc finger for the AT-rich sequence by alpha-helix substitution

In this communication, a novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to that of Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3.2 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.     

Oxidation of Zn(Cys)4 zinc finger peptides by O2 and H2O2: products, mechanism and kinetics

The reactivity of a series of Zn(Cys)(4) zinc finger model peptides towards H(2)O(2) and O(2) has been investigated. The oxidation products were identified by HPLC and ESI-MS analysis. At pH<7.5, the zinc complexes and the free peptides are oxidised to bis-disulfide-containing peptides. Above pH 7.5, the oxidation of the zinc complexes by H(2)O(2) also yields sulfinate- and sulfonate-containing overoxidised peptides. At pH 7.0, monitoring of the reactions between the zinc complexes and H(2)O(2) by HPLC revealed the sequential formation of two disulfides. Several techniques for the determination of the rate constant for the first oxidation step corresponding to the attack of H(2)O(2) by the Zn(Cys)(4) site have been compared. This rate constant can be reliably determined by monitoring the oxidation by HPLC, fluorescence, circular dichroism or absorption spectroscopy in the presence of excess ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid. In contrast, monitoring of the release of zinc with 4-(2-pyridylazo)resorcinol or of the thiol content with 5,5'-dithiobis(2-nitrobenzoate) did not yield reliable values of this rate constant for the case in which the formation of the second disulfide is slower than the formation of the first. The kinetic measurements clearly evidence a protective effect of zinc on the oxidation of the cysteines by both H(2)O(2) and O(2), which points to the fact that zinc binding diminishes the nucleophilicity of the thiolates. In addition, the reaction between the zinc finger and H(2)O(2) is too slow to consider zinc fingers as potential sensors for H(2)O(2) in cells.     

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.     

Targeting retroviral Zn finger-DNA interactions: a small-molecule approach using the electrophilic nature of trans-platinum-nucleobase compounds

Noncovalent interactions are ubiquitous in ternary systems involving metal ions, DNA/RNA, and proteins and represent a structural motif for design of selective inhibitors of biological function. This contribution shows that small molecules containing platinated purine nucleobases mimic the natural DNA(RNA)-tryptophan recognition interaction of zinc finger peptides, specifically the C-terminal finger of HIV NCp7 protein. Interaction with platinum results in Zn ejection from the peptide accompanied by loss of tertiary structure. Targeting the NCp7-DNA interaction for drug design represents a conceptual advance over electrophiles designed for chemical attack on the zinc finger alone. These results demonstrate examples of a new platinum structural class targeting specific biological processes, distinct from the bifunctional DNA-DNA binding of cytotoxic agents like cisplatin. The results confirm the validity of a chemical biological approach for metallodrug design for selective ternary DNA(RNA)-protein interactions.     

Site selection in tandem arrays of metal-binding domains

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical TFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(II) was found to be 15 +/- 10 microM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(II) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.     

Classical Cys2His2 zinc finger peptides are rapidly oxidized by either H2O2 or O2 irrespective of metal coordination

ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.     

Investigation of complexes of DNA with synthetic peptide fragments of the N-end of histone H2B

The nature of the interaction of high- and low-molecular-weight DNAs (6×10⁶ and 3×10⁵ daltons) with synthetic oligopeptides of the N-end of histone H2B having the sequences 1–21, 1–10, 1–13, 11–21, and 14–21, differing in molecular weight and amino acid composition, as a function of the amount of peptide component in the complex and the ionic strength of the solution has been studied by the methods of UV and CD spectroscopy and the spectrophotometric analysis of melting curves. It has been shown that of all the peptides studied only the 1–21 peptide possesses the capacity of condensing DNA. This capacity depends on the amount of peptide component in the complex, the molecular weight of the DNA, and the ionic strength of the solution. The interaction with peptides under all the conditions studied, without changing the conformational parameters of the DNA, stabilizes its secondary structure in relation to the action of the temperature, which depends on the number of lysine residues in the peptides.     

Carboxylic acid functionalized cobalt(III) cyclen complexes for catalytic hydrolysis of phosphodiester bonds

4-(1,4,7,10-Tetraazacyclotetradec-1-yl)methylbenzoic acid (cycmba, 1) has been synthesized, as a step towards the eventual development of sequence-specific hydrolytic complexes. A cobalt(III) complex of 1, [Co(cycmba)Cl2]Cl.1.5H2O (.1.5H2O) was found to be active against both an activated phosphodiester compound, bis(nitrophenyl)phosphate (BNPP), and supercoiled DNA. The presence of the benzoate group depresses the rate of hydrolysis of the ligand-Co(III) system at neutral pH, as confirmed by the kinetics results of a methyl ester analog. The ability of (2.1.5H2O) to bind to solid substrates and remain active was also demonstrated by attachment of the molecule to agarose beads.     

Expanding the DNA-recognition repertoire for zinc finger proteins beyond 20 amino acids

The design of DNA binding domains based on the Cys2His2 zinc finger motif has proven to be a successful strategy for the specific recognition of novel DNA sequences. Although considerable effort has been devoted to the generation of zinc finger proteins with widely varying DNA-binding preferences, only a limited number of potential DNA binding sites have been targeted with a high degree of specificity. These restrictions on zinc finger design appear to be a consequence of the limited repertoire of side-chain lengths and functionalities available with the 20 proteinogenic amino acids. To demonstrate that these limitations can be overcome through the use of "unnatural" amino acids, expressed protein ligation was employed to incorporate the amino acid citrulline into a single position within a three-zinc finger protein. As anticipated, the resulting semisynthetic protein specifically recognizes adenine in the appropriate position of its binding site.     

Investigation of complexes of DNA with synthetic peptide fragments of the N-end of histone H2B

The nature of the interaction of high- and low-molecular-weight DNAs (6×10⁶ and 3×10⁵ daltons) with synthetic oligopeptides of the N-end of histone H2B having the sequences 1–21, 1–10, 1–13, 11–21, and 14–21, differing in molecular weight and amino acid composition, as a function of the amount of peptide component in the complex and the ionic strength of the solution has been studied by the methods of UV and CD spectroscopy and the spectrophotometric analysis of melting curves. It has been shown that of all the peptides studied only the 1–21 peptide possesses the capacity of condensing DNA. This capacity depends on the amount of peptide component in the complex, the molecular weight of the DNA, and the ionic strength of the solution. The interaction with peptides under all the conditions studied, without changing the conformational parameters of the DNA, stabilizes its secondary structure in relation to the action of the temperature, which depends on the number of lysine residues in the peptides.N. I. Nikitin Institute of Chemistry of the TadzhSSR Academy of Sciences, Dushanbe. Institute of Cytology of the USSR Academy of Sciences, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 708–713, September–October, 1987.     

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.     

Sequence and linker dependent chiral dimerization of DNA–porphyrin conjugates

Circular dichroism (CD), UV–vis absorption, fluorescence, and resonance light scattering (RLS) spectroscopies were used to elucidate the role of the DNA sequence, linkers between DNA and porphyrin, and metal in the porphyrin coordination center on the self-assembly of DNA–porphyrin conjugates. A series of eight non-self-complementary DNA–porphyrin conjugates have been synthesized with zinc and free-base porphyrins covalently attached to the short ODNs (A₈ or T₈) via amide or phosphate linker. A small structural modification (e.g., amide linker replaced by the phosphate linker) showed a dramatic effect on the aggregation properties of DNA–porphyrin conjugates and greatly altered their spectroscopic properties. At low ionic strength, porphyrin aggregation was not observed for any conjugate. An increase in the ionic strength caused two out of eight conjugates to form chiral porphyrin dimers.     

FAR UV IRRADIATION OF DNA IN THE PRESENCE OF PROTEINS, AMINO ACIDS OR PEPTIDES

Abstract— The DNA of bacteriophage SP02c12 was subjected to 254 nm irradiation in solutions containing lysozyme or histone. In these solutions, the protein-DNA mass ratios and the ionic strengths of the solvents were varied to change the amount of protein associated with the DNA. Lysozyme-DNA binding constants were measured under the same conditions. The sensitivity of phage DNA to biological inactivation by UV increased as the amount of lysozyme bound per DNA strand increased. Although binding constants could not be measured for the DNA-histone interaction, this protein had a protective effect which was greater under conditions which cause enhanced binding. No crosslinking of either protein could be detected even at doses ten-fold greater than those giving a surviving fraction of 0.01.
Irradiation was also performed in the presence of various amino acids and short peptides. These were chosen to include amino acids which: (1) are positively charged, (2) absorb UV of this wavelength or (3) form UV-induced crosslinks to DNA. None of the amino acids tested affected sensitivity of the DNA to biological inactivation. Peptides containing a UV-absorbing amino acid and a positively charged amino acid enhanced sensitivity. For each of these peptides, a mixture of the constituent amino acids had the same effect as the peptide itself. Under the conditions used, no evidence for formation of DNA-amino acid crosslinks was found. The results indicate that proteins and peptides can sensitize DNA to UV inactivation by mechanisms other than covalent crosslink formation. Such mechanisms could include energy or electron transfer or alterations in the conformation of the DNA.