Metallohistins: a new class of plant metal-binding proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. A reversible shift in histidine C1 and C2 protons in NMR analysis occurred during titration of this protein with ZnCl₂ strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.     

A new class of reverse signaling costimulators belongs to the TNF family

Recent evidence shows that many molecules of the TNF family serve as counter-receptors, inducing costimulation through reverse signals in addition to delivering signals through their respective TNF receptors. In this review, we will discuss this new class of costimulators with a focus on the mechanism of costimulation transduced by reverse signaling through Fas ligand.     

MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism

The outer membrane (OM) of Gram-negative bacteria acts as a formidable barrier against a plethora of detrimental compounds owing to its asymmetric nature. This is because the OM possesses lipopolysaccharides (LPSs) in the outer leaflet and phospholipids (PLs) in the inner leaflet. The maintenance of lipid asymmetry (Mla) system is involved in preserving the distribution of PLs in OM. The periplasmic component of the system MlaC serves as the substrate-binding protein (SBP) that shuttles PLs between the inner and outer membranes. However, an in-depth report highlighting its mechanism of ligand binding is still lacking. This study reports the crystal structure of MlaC from Escherichia coli (EcMlaC) at a resolution of 2.5 Å in a quasi-open state, complexed with PL. The structural analysis reveals that EcMlaC and orthologs comprise two major domains, viz. nuclear transport factor 2-like (NTF2-like) and phospholipid-binding protein (PBP). Each domain can be further divided into two subdomains arranged in a discontinuous fashion. This study further reveals that EcMlaC is polyspecific in nature and follows a reverse mechanism of the opening of the substrate-binding site during the ligand binding. Furthermore, MlaC can bind two PLs by forming subsites in the binding pocket. These findings, altogether, have led to the proposition of the unique “segmented domain movement” mechanism of PL binding, not reported for any known SBP to date. Further, unlike typical SBPs, MlaC has originated from a cystatin-like fold. Overall, this study establishes MlaC to be a non-canonical SBP with a unique ligand-binding mechanism.     

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.     

Glutathione transferases of Phanerochaete chrysosporium: S-glutathionyl-p-hydroquinone reductase belongs to a new structural class

The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.     

A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.     

Fluorescent and photochemical properties of a single zinc finger conjugated to a fluorescent DNA-binding probe

A single zinc finger derived from the DNA-binding domain of the glucocorticoid receptor (GR) has been tethered to the intercalating fluorophore thiazole orange, and the DNA recognition characteristics of the conjugate have been examined. DNA sequence specificity for the peptide-dye conjugate, determined by steady-state fluorescence measurements and photoactivated DNA cleavage experiments, reproduce the binding features of response element recognition found in the native GR. The thiazole orange is able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluorescence is observed from either the conjugate alone or the conjugate mixed with DNA lacking the zinc finger target sequence. The conjugate preferentially targets a 5'-TGTTCT-3' sequence (the native glucocorticoid receptor element) with a dissociation constant of about 25 nM. Lower binding affinities (up to 10-fold) are observed for single site variants of this sequence, and much lower affinity (40-50-fold) is observed for binding to the estrogen response element (which differs from the glucocorticoid receptor element at two positions) as well as to nonspecific DNA. Footprinting reactions show a 4-6 base pair region that is protected by the zinc finger moiety. Photocleavage assays reveal a several base pair region flanking the recognition sequence where the tethered thiazole orange moiety is able to intercalate and subsequently cleave DNA upon visible light exposure. Thiazole orange is also shown to oxidize the 5'-G of remote GG sequences, depending on the details of the intervening DNA sequence. Small synthetic protein-dye conjugates such as this one are potentially useful for a variety of purposes including sequence-specific probes that work under physiological conditions (without melting and hybridization of DNA), sequence-specific photocleavage agents, and self-assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.     

Zinc is required for folding and binding of a single zinc finger to DNA

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non-specifically to DNA only in the presence of zinc. Moreover, competitive DNA binding experiments indicate interaction with 3.9 +/- 0.4 base pairs.     

Towards therapeutic applications of engineered zinc finger proteins

It has long been the goal of molecular biologists to design DNA-binding proteins for the specific control of gene expression. The zinc finger design is ideally suited for such purposes, discriminating between closely related sequences both in vitro and in vivo. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted and switched off and on. Several recent applications of such engineered zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial disease, by stimulating vascular growth. Also in progress are pre-clinical studies using ZFPs to target the defective genes in two monogenic disorders, SCID and SCA. The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of homologous recombination. Promising results are reported.     

Prediction of DNA-binding specificity in zinc finger proteins

Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions -1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure-function relationships of the existing zinc finger-DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.     

Selectivity of arsenite interaction with zinc finger proteins

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ? C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.     

ZFWD: a novel subfamily of plant proteins containing a C3H zinc finger and seven WD40 repeats

We describe a new subfamily of WD repeat proteins characterised by the presence of a C3H zinc finger at the N-terminal part of the protein associated with seven WD40 repeats. We have identified four members of this subfamily in Arabidopsis thaliana, one of them with associated expressed sequence tags (ESTs). We have also identified homologous ESTs in rice, cotton, maize, poplar, pine tree and the ice plant. We do not observe animal homologues, suggesting that this subfamily could be specific for plants. Our data suggest an important role for these proteins. Based on the high sequence conservation within the conserved domains, we suggest that these proteins could have a regulatory function.     

Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.     

Stability of folding structure of Zic zinc finger proteins

Zic family proteins have five C₂H₂-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.