Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

Metal and redox homeostasis in cyanobacteria is tightly controlled to preserve the photosynthetic machinery from mismetallation and minimize cell damage. This control is mainly taken by FUR (ferric uptake regulation) proteins. FurC works as the PerR (peroxide response) paralog in Anabaena sp. PCC7120. Despite its importance, this regulator remained poorly characterized. Although FurC lacks the typical CXXC motifs present in FUR proteins, it contains a tightly bound zinc per subunit. FurC: Zn stoichiometrically binds zinc and manganese in a second site, manganese being more efficient in the binding of FurC: Zn to its DNA target PprxA. Oligomerization analyses of FurC: Zn evidence the occurrence of different aggregates ranging from dimers to octamers. Notably, intermolecular disulfide bonds are not involved in FurC: Zn dimerization, dimer being the most reduced form of the protein. Oligomerization of dimers occurs upon oxidation of thiols by H2O2 or diamide and can be reversed by 1,4-Dithiothreitol (DTT). Irreversible inactivation of the regulator occurs by metal catalyzed oxidation promoted by ferrous iron. However, inactivation upon oxidation with H2O2 in the absence of iron was reverted by addition of DTT. Comparison of models for FurC: Zn dimers and tetramers obtained using AlphaFold Colab and SWISS-MODEL allowed to infer the residues forming both metal-binding sites and to propose the involvement of Cys86 in reversible tetramer formation. Our results decipher the existence of two levels of inactivation of FurC: Zn of Anabaena sp. PCC7120, a reversible one through disulfide-formed FurC: Zn tetramers and the irreversible metal catalyzed oxidation. This additional reversible regulation may be specific of cyanobacteria.

Ligand-Promoted Surface Solubilization of TiO2 Nanoparticles by the Enterobactin Siderophore in Biological Medium.

Titanium dioxide nanoparticles (TiO2-NPs) are increasingly used in consumer products for their particular properties. Even though TiO2 is considered chemically stable and insoluble, studying their behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium-enterobactin (Ti-ent) complex observed. This complex was characterized by UV-visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti-ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO2 food additive in the presence of enterobactin and the entrance of the Ti-enterobactin complex in bacteria were questioned.

Copper-Associated Oxidative Stress Contributes to Cellular Inflammatory Responses in Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF Transmembrane Conductance Regulator (CFTR), an apical chloride channel. An early inflammation (EI) in the lung of CF patients occurring in the absence of any bacterial infection has been reported. This EI has been proposed to be associated with oxidative stress (OX-S), generated by deregulations of the oxidant/antioxidant status. Recently, we demonstrated that copper (Cu), an essential trace element, mediates OX-S in bronchial cells. However, the role of this element in the development of CF-EI, in association with OX-S, has never been investigated. Using healthy (16HBE14o-; HBE), CF (CFBE14o-; CFBE), and corrected-wild type CFTR CF (CFBE-wt) bronchial cells, we characterized the inflammation and OX-S profiles in relation to the copper status and CFTR expression and function. We demonstrated that CFBE cells exhibited a CFTR-independent intrinsic inflammation. These cells also exhibited an alteration in mitochondria, UPR (Unfolded Protein Response), catalase, Cu/Zn- and Mn-SOD activities, and an increase in the intracellular content of iron, zinc, and Cu. The increase in Cu concentration was associated with OX-S and inflammatory responses. These data identify cellular Cu as a key factor in the generation of CF-associated OX-S and opens new areas of investigation to better understand CF-associated EI.

The pathogen Pseudomonas aeruginosa optimizes the production of the siderophore pyochelin upon environmental challenges.

Siderophores are iron chelators produced by bacteria to access iron, an essential nutrient. The pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, the former with a high affinity for iron and the latter with a lower affinity. Furthermore, the production of both siderophores involves a positive auto-regulatory loop: the presence of the ferri-siderophore complex is essential for their large production. Since pyochelin has a lower affinity for iron it was hard to consider the role of pyochelin in drastic competitive environments where the host or the environmental microbiota produce strong iron chelators and may inhibit iron chelation by pyochelin. We showed here that the pyochelin pathway overcomes this difficulty through a more complex regulating mechanism for pyochelin production than previously described. Indeed, in the absence of pyoverdine, and thus higher difficulty to access iron, the bacteria are able to produce pyochelin independently of the presence of ferri-pyochelin. The regulation of the pyochelin pathway appeared to be more complex than expected with a more intricate tuning between repression and activation. Consequently, when the bacteria cannot produce pyoverdine they are able to produce pyochelin even in the presence of strong iron chelators. Such results support a more complex and varied role for this siderophore than previously described, and complexify the battle for iron during P. aeruginosa infection.

Safer-by-design biocides made of tri-thiol bridged silver nanoparticle assemblies.

Silver nanoparticles (AgNPs) are efficient biocides increasingly used in consumer products and medical devices. Their activity is due to their capacity to release bioavailable Ag(i) ions making them long-lasting biocides but AgNPs themselves are usually easily released from the product. Besides, AgNPs are highly sensitive to various chemical environments that triggers their transformation, decreasing their activity. Altogether, widespread use of AgNPs leads to bacterial resistance and safety concerns for humans and the environment. There is thus a crucial need for improvement. Herein, a proof of concept for a novel biocide based on AgNP assemblies bridged together by a tri-thiol bioinspired ligand is presented. The final nanomaterial is stable and less sensitive to chemical environments with AgNPs completely covered by organic molecules tightly bound via their thiol functions. Therefore, these AgNP assemblies can be considered as safer-by-design and innovative biocides, since they deliver a sufficient amount of Ag(i) for biocidal activity with no release of AgNPs, which are insensitive to transformations in the nanomaterial.

Non-specific interference of cobalt with siderophore-dependent iron uptake pathways.

Much data shows that biological metals other than Fe3+ can interfere with Fe3+ acquisition by siderophores in bacteria. Siderophores are small Fe3+ chelators produced by the microorganisms to obtain access to Fe3+. Here, we show that Co2+ is imported into Pseudomonas aeruginosa cells in a complex with the siderophore pyochelin (PCH) by the ferri-PCH outer membrane transporter FptA. Moreover, the presence of Co2+ in the bacterial environment strongly affects the production of PCH. Proteomic and transcriptomic approaches showed that a decrease of PCH production is associated with repression of the expression of the genes involved in PCH biosynthesis. We used various molecular biology approaches to show that this repression is not Fur-(ferric uptake transcriptional regulator) dependent but due to competition of PCH-Co with PCH-Fe for PchR (transcriptional activator), thus inhibiting the formation of PchR-PCH-Fe and consequently the expression of the PCH genes. We observed a similar mechanism of repression of PCH production, but to a lesser extent, by Ni2+, but not for Zn2+, Cu2+, or Mn2+. Here, we show, for the first time at a molecular level, how the presence of a contaminant metal can interfere with Fe3+ acquisition by the siderophores PCH and PVD.

The role of cysteine and sulfide in the interplay between microbial Hg(ii) uptake and sulfur metabolism.

Biogenic thiols, such as cysteine, have been used to control the speciation of Hg(ii) in bacterial exposure experiments. However, the extracellular biodegradation of excess cysteine leads to the formation of Hg(ii)-sulfide species, convoluting the interpretation of Hg(ii) uptake results. Herein, we test the hypothesis that Hg(ii)-sulfide species formation is a critical step during bacterial Hg(ii) uptake in the presence of excess cysteine. An Escherichia coli (E. coli) wild-type and mutant strain lacking the decR gene that regulates cysteine degradation to sulfide were exposed to 50 and 500 nM Hg with 0 to 2 mM cysteine. The decR mutant released ∼4 times less sulfide from cysteine degradation compared to the wild-type for all tested cysteine concentrations during a 3 hour exposure period. We show with thermodynamic calculations that the predicted concentration of Hg(ii)-cysteine species remaining in the exposure medium (as opposed to forming HgS(s)) is a good proxy for the measured concentration of dissolved Hg(ii) (i.e., not cell-bound). Likewise, the measured cell-bound Hg(ii) correlates with thermodynamic calculations for HgS(s) formation in the presence of cysteine. High resolution X-ray absorption near edge structure (HR-XANES) spectra confirm the existence of cell-associated HgS(s) at 500 nM total Hg and suggest the formation of Hg-S clusters at 50 nM total Hg. Our results indicate that a speciation change to Hg(ii)-sulfide controls Hg(ii) cell-association in the presence of excess cysteine.

Involvement of the Prion Protein in the Protection of the Human Bronchial Epithelial Barrier Against Oxidative Stress.

Aim: Bronchial epithelium acts as a defensive barrier against inhaled pollutants and microorganisms. This barrier is often compromised in inflammatory airway diseases that are characterized by excessive oxidative stress responses, leading to bronchial epithelial shedding, barrier failure, and increased bronchial epithelium permeability. Among proteins expressed in the junctional barrier and participating to the regulation of the response to oxidative and to environmental stresses is the cellular prion protein (PrPC). However, the role of PrPC is still unknown in the bronchial epithelium. Herein, we investigated the cellular mechanisms by which PrPC protein participates into the junctional complexes formation, regulation, and oxidative protection in human bronchial epithelium. Results: Both PrPC messenger RNA and mature protein were expressed in human epithelial bronchial cells. PrPC was localized in the apical domain and became lateral, at high degree of cell polarization, where it colocalized and interacted with adherens (E-cadherin/γ-catenin) and desmosomal (desmoglein/desmoplakin) junctional proteins. No interaction was detected with tight junction proteins. Disruption of such interactions induced the loss of the epithelial barrier. Moreover, we demonstrated that PrPC protection against copper-associated oxidative stress was involved in multiple processes, including the stability of adherens and desmosomal junctional proteins. Innovation: PrPC is a pivotal protein in the protection against oxidative stress that is associated with the degradation of adherens and desmosomal junctional proteins. Conclusion: Altogether, these results demonstrate that the loss of the integrity of the epithelial barrier by oxidative stress is attenuated by the activation of PrPC expression, where deregulation might be associated with respiratory diseases.

Textural, Structural and Biological Evaluation of Hydroxyapatite Doped with Zinc at Low Concentrations.

The present work was focused on the synthesis and characterization of hydroxyapatite doped with low concentrations of zinc (Zn:HAp) (0.01 < xZn < 0.05). The incorporation of low concentrations of Zn2+ ions in the hydroxyapatite (HAp) structure was achieved by co-precipitation method. The physico-chemical properties of the samples were characterized by X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning Electron Microscopy (SEM), zeta-potential, and DLS and N2-BET measurements. The results obtained by XRD and FTIR studies demonstrated that doping hydroxyapatite with low concentrations of zinc leads to the formation of a hexagonal structure with lattice parameters characteristic to hydroxyapatite. The XRD studies have also shown that the crystallite size and lattice parameters of the unit cell depend on the substitutions of Ca2+ with Zn2+ in the apatitic structure. Moreover, the FTIR analysis revealed that the water content increases with the increase of zinc concentration. Furthermore, the Energy Dispersive X-ray Analysis (EDAX) and XPS analyses showed that the elements Ca, P, O, and Zn were found in all the Zn:HAp samples suggesting that the synthesized materials were zinc doped hydroxyapatite, Ca10-xZnx(PO4)6(OH), with 0.01 ≤ xZn ≤ 0.05. Antimicrobial assays on Staphylococcus aureus and Escherichia coli bacterial strains and HepG2 cell viability assay were carried out.

Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(i), corona formation and enzymatic activity.

The release of Ag(i) from silver nanoparticles (AgNPs) unintentionally spread in the environment is suspected to impair some key biological functions. In comparison with AgNO3, in-depth investigations were carried out into the interactions between citrate-coated AgNPs (20 nm) and two metalloproteins, intracellular metallothionein 1 (MT1) and plasmatic ceruloplasmin (Cp), both involved in metal homeostasis. These were chosen for their physiological relevance and the diversity of their various native metals bound because of thiol groups and/or their structural differences. Transmission electron microscopy (TEM), and dynamic light scattering (DLS), UV-vis and circular dichroism (CD) spectroscopies were used to study the effects of such intricate interactions on AgNP dissolution and proteins in terms of metal exchanges and structural modifications. The isolation of the different populations formed together with on-line quantifications of their metal content were performed by asymmetrical flow field-flow fractionation (AF4) linked to inductively coupled plasma mass spectrometry (ICP-MS). For the 2 proteins, Ag(i) dissolved from the AgNPs, substituted for the native metal, to different extents and with different types of dynamics for the corona formed: the MT1 rapidly surrounded the AgNPs with the transient reticulate corona thus promoting their dissolution associated with the metal substitution, whereas the Cp established a more stable layer around the AgNPs, with a limited substitution of Cu and a decrease in its ferroxidase activity. The accessibility and lability of the metal binding sites inside these proteins and their relative affinities for Ag(i) are discussed, taking into account the structural characteristics of the proteins.

Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging, synthesis and applications.

BACKGROUND: The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo. SCOPE OF REVIEW: In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs. MAJOR CONCLUSIONS: Labile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity. GENERAL SIGNIFICANCE: The exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context.

From Peptide Aptamers to Inhibitors of FUR, Bacterial Transcriptional Regulator of Iron Homeostasis and Virulence.

FUR (Ferric Uptake Regulator) protein is a global transcriptional regulator that senses iron status and controls the expression of genes involved in iron homeostasis, virulence, and oxidative stress. Ubiquitous in Gram-negative bacteria and absent in eukaryotes, FUR is an attractive antivirulence target since the inactivation of the fur gene in various pathogens attenuates their virulence. The characterization of 13-aa-long anti-FUR linear peptides derived from the variable part of the anti-FUR peptide aptamers, that were previously shown to decrease pathogenic E. coli strain virulence in a fly infection model, is described herein. Modeling, docking, and experimental approaches in vitro (activity and interaction assays, mutations) and in cells (yeast two-hybrid assays) were combined to characterize the interactions of the peptides with FUR, and to understand their mechanism of inhibition. As a result, reliable structure models of two peptide-FUR complexes are given. Inhibition sites are mapped in the groove between the two FUR subunits where DNA should also bind. Another peptide behaves differently and interferes with the dimerization itself. These results define these novel small peptide inhibitors as lead compounds for inhibition of the FUR transcription factor.

Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins.

The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites.

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination number, with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, respectively, comparable to the values reported for crystalline Ag(I)-thiolate compounds. The bioinspired Cu(I) chelator L(1) is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a reference compound for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the average Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, respectively, both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chemistry of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.

Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches.

The FUR protein (ferric uptake regulator) is an iron-dependent global transcriptional regulator. Specific to bacteria, FUR is an attractive antibacterial target since virulence is correlated to iron bioavailability. Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable loops inserted into a thioredoxinA scaffold, were identified, which were able to interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to decrease the virulence of pathogenic E. coli in a fly infection model. The first characterization of anti-FUR linear peptides (pF1 6 to 13 amino acids) derived from the variable part of the F1 anti-FUR peptide aptamer is described herein. Theoretical and experimental approaches, in original combination, were used to study interactions of these peptides with FUR in order to understand their mechanism of inhibition. After modeling EcFUR by homology, docking with Autodock was combined with molecular dynamics simulations in implicit solvent to take into account the flexibility of the partners. All calculations were cross-checked either with other programs or with experimental data. As a result, reliable structures of EcFUR and its complex with pF1 are given and an inhibition pocket formed by the groove between the two FUR subunits is proposed. The location of the pocket was validated through experimental mutation of key EcFUR residues at the site of proposed peptide interaction. Cyclisation of pF1, mimicking the peptide constraint in F1, improved inhibition. The details of the interactions between peptide and protein were analyzed and a mechanism of inhibition of these anti-FUR molecules is proposed.

Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions.

Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu overload, a Cu-Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.

Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori.

Nickel is an essential metal for Helicobacter pylori, as it is the co-factor of two enzymes crucial for colonization, urease and hydrogenase. Nickel is taken up by specific transporters and its intracellular homeostasis depends on nickel-binding proteins to avoid toxicity. Nickel trafficking is controlled by the Ni(II)-dependent transcriptional regulator NikR. In contrast to other NikR proteins, NikR from H. pylori is a pleiotropic regulator that depending on the target gene acts as an activator or a repressor. We systematically quantified the in vivo Ni(2+)-NikR response of 11 direct NikR targets that encode functions related to nickel metabolism, four activated and seven repressed genes. Among these, four targets were characterized for the first time (hpn, hpn-like, hydA and hspA) and NikR binding to their promoter regions was demonstrated by electrophoretic mobility shift assays. We found that NikR-dependent repression was generally set up at higher nickel concentrations than activation. Kinetics of the regulation revealed a gradual and temporal NikR-mediated response to nickel where activation of nickel-protection mechanisms takes place before repression of nickel uptake. Our in vivo study demonstrates, for the first time, a chronological hierarchy in the NikR-dependent transcriptional response to nickel that is coherent with the control of nickel homeostasis in H. pylori.

The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.

Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS4 site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

A ZnS(4) structural zinc site in the Helicobacter pylori ferric uptake regulator.

The ferric uptake regulator, Fur, is a global bacterial transcriptional regulator using iron as a cofactor to bind to specific DNA sequences. This paper describes the biochemical characterization of the native ferric uptake regulator from Helicobacter pylori (HpFur): oligomeric state, metal content, and characterization of a structural metal-binding site. HpFur contains six cysteines with two CxxC motifs, which makes it closer to Bacillus subtilis PerR (BsPerR) than to Escherichia coli Fur (EcFur). Chemical modifications of cysteine residues using iodoacetamide followed by mass spectrometry after enzymatic digestion strongly suggest that these two CxxC motifs containing cysteines 102-105 and 142-145 are involved in zinc binding in a ZnS(4) metal site. The other two cysteines (78 and 150) are not essential for DNA binding activity and do not perturb metal binding as demonstrated with the characterization of a FurC78SC150S double mutant. Chelating agent such as EDTA disrupts the dimeric structure into monomer which did not contain zinc anymore. Reconstitution of dimer from monomer requires reduction and Zn(2+) binding. Cadmium(II) substitution allows also dimer formation from monomer, and Cd(II)-substituted FurC78SC150S mutant presents a characteristic absorption of a Cd(II)Cys(4) metal-binding site. These results establish that coordination of the zinc ion in HpFur is ZnCys(4), therefore closer to the zinc site in BsPerR than in EcFur. Furthermore, the redox state of the cysteines and the zinc binding are essential to hold the H. pylori Fur in a dimeric state.

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.