Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> topoisomerase I is an iron and zinc binding protein

Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV–Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.     

Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD

YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.     

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.     

ZNF300, a recently identified human transcription factor,activates the human IL-2Rβ promoter through the overlapping ZNF300/EGR1 binding site

ZNF300 was recently identified as a member of the human KRAB/C₂H₂ zinc finger protein family. Little is known about the role of ZNF300 in human gene regulation networks. In this study, the DNA-binding property of ZNF300 was further analyzed. We found that the recombinant ZNF300 could bind to the binding site 5′-GCGGGGGCG-3′ of Egr1, another member of the KRAB/C₂H₂ zinc finger protein family. Similarly, recombinant Egr1 also showed a similar binding affinity to the ZNF300 binding site 5′-CTGGGGGCG-3′. Bioinformatics analysis revealed that there is an overlapping ZNF300/Egr1 binding site in the human IL-2Rβ promoter region, which was previously known to be recognized by endogenous Egr1. Electrophoretic mobility shift assays showed that endogenous ZNF300 could also bind to this site. A transient transfection assay revealed that both ZNF300 and Egr1 could transactivate the IL-2Rβ promoter, and that the activation was abrogated by a mutation of residues in the overlapping ZNF300/Egr1 binding site. Co-expression of ZNF300 and Egr1 led to enhanced IL-2Rβ promoter activity. Thus, ZNF300 is likely to be another regulator of the human IL-2Rβ promoter.     

Air-stable,heme-like water-soluble iron(II) porphyrin: in situ preparation and characterization

Preparation of the water-soluble, kinetically labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)porphyrin, Fe(II)TPPS⁴⁻, has been realized in neutral or weakly acidic solutions containing acetate buffer. The buffer played a double role in these systems: it was used for both adjusting pH and, via formation of an acetato complex, trapping trace amounts of iron(III) ions, which would convert the iron(II) porphyrins to the corresponding iron(III) species. Fe(II)TPPS⁴⁻ proved to be stable in these solutions even after saturation with air or oxygen. In the absence of acetate ions, however, iron(II) ions play a catalytic role in the formation of iron(III) porphyrins. While the kinetically inert iron(III) porphyrin, Fe(III)TPPS³⁻, is a regular one with no emission and photoredox properties, the corresponding iron(II) porphyrin displays photoinduced features which are typical of sitting-atop complexes (redshifted Soret absorption and blueshifted emission and Q absorption bands, photoinduced porphyrin ligand-to-metal charge transfer, LMCT, reaction). In the photolysis of Fe(II)TPPS⁴⁻ the LMCT process is followed by detachment of the reduced metal center and an irreversible ring-opening of the porphyrin ligand, resulting in the degradation of the complex. Possible oxygen-binding ability of Fe(II)TPPS⁴⁻ (as a heme model) has been studied as well. Density functional theory calculations revealed that in solutions with high acetate concentration there is very little chance for iron(II) porpyrin to bind and release O₂, deviating from heme in a hydrophobic microenvironment in hemoglobin. In the presence of an iron(III)-trapping additive that is much less strongly coordinated to the iron(II) center than the acetate ion, Fe(II)TPPS⁴⁻ may function as a heme model.     

Isolation and characterization of a new gene, sre , which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ⁻ strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ⁻ mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells. Received: 20 January 1998 / Accepted: 23 April 1998     

Associations among plasma selenium,zinc, copper,and iron concentrations and immunoregulatory cytokine levels in patients with cutaneous leishmaniasis

Plasma essential trace elements, selenium, copper, zinc, and iron concentrations and the levels of immunoregulatory cytokines, interleukin-1β (IL-1β), interleukin-2 receptor (IL-2r), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were evaluated in patients with cutaneous leishmaniasis (CL) to investigate a possible role of these cytokines on selenium, zinc, copper, and iron homeostasis in CL patients. Plasma albumin levels were measured as an index of nutritional status. Plasma selenium, zinc, and iron concentrations, and IL-2r levels were significantly lower, and copper concentrations and IL-1β, IL-8, IL-6 and TNF-α levels were significantly higher in patients with CL than those of healthy controls. There was no significant difference in plasma albumin levels between two groups. There were positive important correlations between plasma selenium and IL-2r, copper and IL-6, and copper and IL-1β, and negative correlations between selenium and IL-8, iron and TNF-α, and zinc and IL-1β contents in patients with CL. Our results showed that plasma trace element contents change in patients with CL. These changes may not be a result of a specific deficiency from dietary inadequacies or imbalances, but, probably, a result of a part of the defense strategies of an organism that is regulated by immunoregulatory cytokines.     

The Arabidopsis TAG1 transposase has an N-terminal zinc finger DNA binding domain that recognizes distinct subterminal motifs

The in vitro DNA binding activity of the Arabidopsis Tag1 transposase (TAG1) was characterized to determine the mechanism of DNA recognition. In addition to terminal inverted repeats, the Tag1 element contains four different subterminal repeats that flank a transcribed region encoding a 729-amino acid protein. A single site-specific DNA binding domain is located near the N terminus of TAG1, between residues 21 and 133. This domain binds specifically to the AAACCC and TGACCC subterminal repeats, found near the 5' and 3' ends of the element, respectively. The ACCC sequence within these repeats is critical for recognition because mutations at positions 3, 5, and 6 abolished binding, yet the first two bases also are important because substitutions at these positions decreased binding by up to 90%. Weak interaction also occurs with the terminal inverted repeats, but no binding was observed to the other two 3' subterminal repeat regions. Sequence analysis of the TAG1 DNA binding domain revealed a C(2)HC zinc finger motif. Tests for metal dependence showed that DNA binding activity was inhibited by divalent metal chelators and greatly enhanced by zinc. Furthermore, mutation of each cysteine residue predicted to be a metal ligand in the C(2)HC motif abolished DNA binding. Together, these data show that the DNA binding domain of TAG1 specifically binds to distinct subterminal repeats and contains a zinc finger.