Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.     

Dps/Dpr ferritin-like protein: insights into the mechanism of iron incorporation and evidence for a central role in cellular iron homeostasis in Streptococcus suis

The Dps family members constitute a distinct group of multimeric and ferritin-like iron binding proteins (up to 500 iron atoms/12-mer) that are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge, the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen Streptococcus suis that: (i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III); (ii) Fe(II) atoms enter the 12-mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12-mer cavity at 12 identical inter-subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the Dps/Dpr ferritin-like protein is proposed.     

Crystal structure of Streptococcus suis Dps-like peroxide resistance protein Dpr: implications for iron incorporation

The Dps-like peroxide resistance protein (Dpr) is an aerotolerance and hydrogen peroxide resistance agent found in the meningitis-associated pathogen Streptococcus suis. Dpr is believed to act by binding free intracellular iron to prevent Fenton chemistry-catalysed formation of toxic hydroxyl radicals. The crystal structure of Dpr has been determined to 1.95 A resolution. The final model has an Rcyst value of 18.5% (Rfree = 22.4%) and consists of 12 identical monomers (each of them comprising a four alpha-helix bundle) that form a hollow sphere obeying 23 symmetry. Structural features show that Dpr belongs to the Dps family of bacterial proteins. Twelve putative ferroxidase centers, each formed at the interface of neighboring monomer pairs, were identified in the Dpr structure with structural similarities to those found in other Dps family members. Dpr was crystallized in the absence of iron, hence no bound iron was found in the structure in contrast to other Dps family members. A novel metal-binding site approximately 6A from the ferroxidase centre was identified and assigned to a bound calcium ion. Two residues from the ferroxidase centre (Asp63 and Asp74) were found to be involved in calcium binding. Structural comparison with other family members revealed that Asp63 and Asp74 adopt different conformation in the Dpr structure. The structure of Dpr presented here shows potential local conformational changes that may occur during iron incorporation. A role for the metal-binding site in iron uptake is proposed.     

An iron-binding protein,Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro

The dpr gene is an antioxidant gene which was isolated from the Streptococcus mutans chromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant of Escherichia coli, and it was proven to play an indispensable role in oxygen tolerance in S. mutans. Here, we purified the 20-kDa dpr gene product, Dpr, from a crude extract of S. mutans as an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. Unlike E. coli Dps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 microM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein in S. mutans and may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.     

Crystal structures of Streptococcus pyogenes Dpr reveal a dodecameric iron-binding protein with a ferroxidase site

DNA-binding protein from starved cells (Dps)-like proteins are key factors involved in oxidative stress protection in bacteria. They bind and oxidize iron, thus preventing the formation of harmful reactive oxygen species that can damage biomolecules, particularly DNA. Dps-like proteins are composed of 12 identical subunits assembled in a spherical structure with a hollow central cavity. The iron oxidation occurs at 12 intersubunit sites located at dimer interfaces. Streptococcus pyogenes Dps-like peroxide resistance protein (Dpr) has been previously found to protect the catalase-lacking S. pyogenes bacterium from oxidative stress. We have determined the crystal structure of S. pyogenes Dpr, the second Dpr structure from a streptococcal bacterium, in iron-free and iron-bound forms at 2.0- and 1.93-Å resolution, respectively. The iron binds to well-conserved sites at dimer interfaces and is coordinated directly to Asp77 and Glu81 from one monomer, His50 from a twofold symmetry-related monomer, a glycerol molecule, and a water molecule. Upon iron binding, Asp77 and Glu81 change conformation. Site-directed mutagenesis of active-site residues His50, His62, Asp66, Asp77, and Glu81 to Ala revealed a dramatic decrease in iron incorporation. A short helix at the N-terminal was found in a different position compared with other Dps-like proteins. Two types of pores were identified in the dodecamer. Although the N-terminal pore was found to be similar to that of other Dps-like proteins, the C-terminal pore was found to be blocked by bulky Tyr residues instead of small residues present in other Dps-like proteins.     

Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis

H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.     

Iron content of Streptococcus suis and evidence for a dpr homologue

The type strain of Streptococcus suis was investigated for features that might help the organism to tolerate the H2O2 that is produced during growth. Enzyme assays, using soluble extracts, revealed that the type strain, which lacks catalase, lacks NADH peroxidase in both the mid-exponential and stationary phases of the growth cycle. Although iron could not be detected colourimetrically in dense cell suspensions, determination of the cellular iron content following growth to early stationary phase in the presence of 55FeCl3 demonstrated that S. suis does contain iron and hence is incapable of iron exclusion. Gene amplification, using oligonucleotide primers based on dpr of Streptococcus mutans, followed by nucleotide sequencing, revealed in S. suis, the presence of a gene that encodes a Dpr homologue. It is concluded that in S. suis, tolerance of H2O2 is due to iron sequestration by Dpr and the consequent effect of this process on the extent of Fenton chemistry.     

Overexpression and characterization of an iron storage and DNA-binding Dps protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N(2)-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps(T. erythraeurm) protein (Dps(tery)) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps(tery), and we found that Dps(tery), like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps(tery) binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps(tery) indicated that it has an iron core that resembles that of horse spleen ferritin.     

The crystal structure of Deinococcus radiodurans Dps protein (DR2263) reveals the presence of a novel metal centre in the N terminus

The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-Å resolution, and one soaked in an iron solution, to 1.6-Å resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Magnetic properties and structural characterization of iron oxide nanoparticles formed by <Emphasis Type="Italic">Streptococcus suis</Emphasis> Dpr and four mutants

Streptococcus suis Dpr belongs to the Dps family of bacterial and archaeal proteins that oxidize Fe²⁺ to Fe³⁺ to protect microorganisms from oxidative damage. The oxidized iron is subsequently deposited as ferrihydrite inside a protein cavity, resulting in the formation of an iron core. The size and the magnetic properties of the iron core have attracted considerable attention for nanotechnological applications in recent years. Here, the magnetic and structural properties of the iron core in wild-type Dpr and four cavity mutants were studied. All samples clearly demonstrated a superparamagnetic behavior in superconducting quantum interference device magnetometry and Mössbauer spectroscopy compatible with that of superparamagnetic ferrihydrite nanoparticles. However, all the mutants exhibited higher magnetic moments than the wild-type protein. Furthermore, measurement of the iron content with inductively coupled plasma mass spectrometry revealed a smaller amount of iron in the iron cores of the mutants, suggesting that the mutations affect nucleation and iron deposition inside the cavity. The X-ray crystal structures of the mutants revealed no changes compared with the wild-type crystal structure; thus, the differences in the magnetic moments could not be attributed to structural changes in the protein. Extended X-ray absorption fine structure measurements showed that the coordination geometry of the iron cores of the mutants was similar to that of the wild-type protein. Taken together, these results suggest that mutation of the residues that surround the iron storage cavity could be exploited to selectively modify the magnetic properties of the iron core without affecting the structure of the protein and the geometry of the iron core.     

Bacterial zinc uptake and regulators

Many bacteria use an ABC transporter for high-affinity uptake of zinc with a cluster 9 solute-binding protein. Other members of this protein family transport manganese. At present, it is not always possible to distinguish zinc-specific and manganese-specific transporters on the basis of sequence analysis. Low-affinity ZIP-type zinc transporters in bacteria have also been identified. Most high-affinity zinc uptake systems are regulated by Zur proteins, which form at least three unrelated subgroups of the Fur protein family (regulators of iron transport). High-affinity transport of zinc out of the periplasmic space poses a problem to the cell because zinc is a cofactor of several periplasmic enzymes. Certain zinc-binding proteins in the periplasm might function as chaperones to supply these enzymes with zinc.     

Co-expression of zinc binding motif and GFP as a cellular indicator of metal ions mobility

A significant role of zinc-binding motifs on metal mobility in Escherichia coli was explored using a chimeric metal-binding green fluorescent protein (GFP) as an intracellular zinc indicator. Investigation was initiated by co-transformation and co-expression of two chimeric genes encoding the chimeric GFP carrying hexahistidine (His6GFP) and the zinc-binding motif fused to outer membrane protein A (OmpA) in E. coli strain TG1. The presence of these two genes was confirmed by restriction endonucleases analysis. Co-expression of the two recombinant proteins exhibited cellular fluorescence activity and enhanced metal-binding capability of the engineered cells. Incorporation of the zinc-binding motif onto the membrane resulted in 60-fold more binding capability to zinc ions than those of the control cells. The high affinity to metal ions of the bacterial surface influenced influx of metal ions to the cells. This may affect the essential ions for triggering important cell metabolism. A declining of fluorescent intensity of GFP has been detected on the cell expressed of zinc binding motif. Meanwhile, balancing of metal homeostasis due to the presence of cytoplasmic chimeric His6GFP enhanced the fluorescent emission. These findings provide the first evidence of real-time monitoring of intracellular mobility of zinc by autofluorescent proteins.     

Dpr蛋白在寡发酵链球菌抗过氧化氢中的作用

摘要：【目的】寡发酵链球菌（Streptococcus oligofermentans）是从无龋人的口腔中分离到的一株链球菌,好氧条件下产生、同时也耐受高浓度（4.4 mmol/L）的过氧化氢。本研究探讨dpr基因对寡发酵链球菌抗过氧化氢的贡献。【方法】克隆和表达寡发酵链球菌dpr基因,分析Dpr蛋白的功能;构建寡发酵链球菌的dpr基因突变株,比较野生株和突变株对不同浓度过氧化氢的耐受程度;并将寡发酵链球菌dpr基因克隆到对过氧化氢耐受力低的变形链球菌中,分析其对变形链球菌过氧化氢耐受能力的影响。【结果】     

The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site

The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.     

Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn(2+)- or Cd(2+)-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.     

Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N₂-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps_{T. erythraeurm} protein (Dps_tery) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps_tery, and we found that Dps_tery, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps_tery binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps_tery indicated that it has an iron core that resembles that of horse spleen ferritin.     

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.     

The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni

We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H(2)O(2) or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.