DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus

Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress. The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively. Gel mobility and atomic force microscopy imaging showed that at pH 6.3, both wild type and DpsDelta8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes. Conversely, DpsDelta18 does not self-aggregate and binds DNA without causing condensation. At pH 8.2, DpsDelta8 and DpsDelta18 neither self-aggregate nor cause DNA condensation, a behavior also displayed by wild-type Dps at pH 8.7. Thus, Dps self-aggregation and Dps-driven DNA condensation are parallel phenomena that reflect the properties of the N-terminus. DNA protection against the toxic action of Fe(II) and H2O2 is not affected by the N-terminal deletions either in vitro or in vivo, in accordance with the different structural basis of this property.     

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.     

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.     

One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

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.     

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 multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding

Background

The widely expressed Dps proteins, so named after the DNA-binding properties of the first characterized member of the family in Escherichia coli, are considered major players in the bacterial response to stress.

Scope of review

The review describes the distinctive features of the “ferritin-like” ferroxidation reaction, which uses hydrogen peroxide as physiological iron oxidant and therefore permits the concomitant removal of the two reactants that give rise to hydroxyl radicals via Fenton chemistry. It also illustrates the structural elements identified to date that render the interaction of some Dps proteins with DNA possible and outlines briefly the significance of Dps–DNA complex formation and of the Dps interaction with other DNA-binding proteins in relation to the organization of the nucleoid and microbial survival.

General significance

Understanding in molecular terms the distinctive role of Dps proteins in bacterial resistance to general and specific stress conditions.

Major conclusions

The state of the art is that the response to oxidative and peroxide-mediated stress is mediated directly by Dps proteins via their ferritin-like activity. In contrast, the response to other stress conditions derives from the concerted interplay of diverse interactions that Dps proteins may establish with DNA and with other DNA-binding proteins.     

The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure,iron binding,and hydroxyl-radical scavenging properties

Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H2O2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H2O2, the major component of the plant defense system when challenged by the bacterium.     

Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA

Ferritin-like proteins, Dps (DNA-binding protein from starved cells), store iron and play a key role in the iron homeostasis in bacteria, yet their iron releasing machinery remains largely unexplored. The electron donor proteins that may interact with Dps and promote the mobilization of the stored iron have hitherto not been identified. Here, we investigate the binding capacity of the two atypical Dps proteins NpDps4 and NpDps5 from Nostoc punctiforme to isolated ferredoxins. We report NpDps-ferredoxin interactions by fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) methods. Dynamic light scattering, size exclusion chromatography and native gel electrophoresis results show that NpDps4 forms a dodecamer at both pH 6.0 and pH 8.0, while NpDps5 forms a dodecamer only at pH 6.0. In addition, FCS data clearly reveal that the non-canonical NpDps5 interacts with DNA at pH 6.0. Our spectroscopic analysis shows that [FeS] centers of the three recombinantly expressed and isolated ferredoxins are properly incorporated and are consistent with their respective native states. The results support our hypothesis that ferredoxins could be involved in cellular iron homeostasis by interacting with Dps and assisting the release of stored iron.     

Reassessment of protein stability, DNA binding, and protection of Mycobacterium smegmatis Dps

The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji, in which the C terminus that is used for binding DNA contains a histidine tag, have been characterized in parallel. The native dodecamer dissociated reversibly into dimers above pH 7.5 and below pH 6.0, with apparent pK(a) values of approximately 7.65 and 4.75; at pH approximately 4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu(157) and Arg(99) located at the 3-fold symmetry axes and to protonation of Asp(66) hydrogen-bonded to Lys(36) across the dimer interface, respectively. The C-terminal tag did not affect subunit dissociation, but altered DNA binding dramatically. At neutral pH, protonation of the histidine tag promoted DNA condensation, whereas in the native C terminus, compensation of negative and positive charges led to DNA binding without condensation. This different mode of interaction with DNA has important functional consequences as indicated by the failure of the native protein to protect DNA from DNase-mediated cleavage and by the efficiency of the tagged protein in doing so as a result of DNA sequestration in the condensates. Chemical protection of DNA from oxidative damage is realized by Dps proteins in a multistep iron oxidation/uptake/mineralization process. Dimers have a decreased protection efficiency due to disruption of the dodecamer internal cavity, where iron is deposited and mineralized after oxidation at the ferroxidase center.     

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.     

Differential DNA binding and protection by dimeric and dodecameric forms of the ferritin homolog Dps from Deinococcus radiodurans

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D.radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(+/-0.1) degrees C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca(2+) (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry.     

Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus

DNA-binding proteins from starved cells (Dps proteins) protect bacteria primarily from oxidative damage. They are composed of 12 identical subunits assembled with 23-symmetry to form a compact cage-like structure known to be stable at temperatures > 70 degrees C and over a wide pH range. Thermosynechococcus elongatus Dps thermostability is increased dramatically relative to mesophilic Dps proteins. Hydrophobic interactions at the dimeric and trimeric interfaces called Dps-like are replaced by salt bridges and hydrogen bonds, a common strategy in thermophiles. Moreover, the buried surface area at the least-extended Dps-like interface is significantly increased. A peculiarity of T. elongatus Dps is the presence of a chloride ion coordinated with threefold symmetry-related arginine residues lining the opening of the Dps-like pore toward the internal cavity. T. elongatus Dps conserves the unusual intersubunit ferroxidase centre that allows the Dps protein family to oxidize Fe(II) with hydrogen peroxide, thereby inhibiting free radical production via Fenton chemistry. This catalytic property is of special importance in T. elongatus (which lacks the catalase gene) in the protection of DNA and photosystems I and II from hydrogen peroxide-mediated oxidative damage.     

Helicobacter hepaticus Dps protein plays an important role in protecting DNA from oxidative damage

The ferritin-like DNA-binding protein from starved cells (Dps) family proteins are present in a number of pathogenic bacteria. Dps in the enterohepatic pathogen, Helicobacter hepaticus is characterized and a H. hepaticus dps mutant was generated by insertional mutagenesis. While the wild type H. hepaticus cells were able to survive in an atmosphere containing up to 6.0% O₂, the dps mutant failed to grow in 3.0% O₂, and it was also more sensitive to oxidative reagents like H₂O₂, cumene hydroperoxide and t-butyl hydroperoxide. Upon air exposure, the dps^-  cells had more damaged DNA than the wild type; they became coccoid or lysed and they contained ∼6-fold higher amount of 8-oxoguanine (8-oxoG) DNA lesions than wild type cells. Purified H. hepaticus Dps was shown to be able to bind both iron and DNA. The iron-loaded form of Dps protein had much greater DNA binding ability than the native Dps or the iron-free Dps.     

Expression and mutagenesis of the dpsA gene of Synechococcus sp. PCC7942, encoding a DNA-binding protein involved in oxidative stress protection

The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long-term nutrient limitation. Some members of the Dps protein family have been shown to form abundant, large (∼150 kD) hexameric complexes that bind chromosomal DNA with little sequence specificity. Previous work from this lab has demonstrated that the Dps proteins are divergent members of the bacterioferritin/bacterioferritin superfamily, and that the Synechococcus sp. PCC7942 Dps homolog, named DpsA, is a DNA-binding hemoprotein having heme-dependent catalytic activity. We speculated that this protein may yield a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggested that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Current work has examined the expression of dpsA both under nutrient stress and during the growth phase; whereas dpsA mRNA is detectable in the exponential phase, transition to stationary phase yields a 20-fold increase in steady-state mRNA levels. Mapping the promoter region identifies a TAGAAT −10 sequence likely recognized by a cyanobacterial RpoS homolog. Lastly, site-directed mutants lacking dpsA function exhibit a severe phenotype impaired under all conditions yielding photooxidative stress; these include high light and treatment with paraquat. This supports our contention that the DpsA protein serves an important protective function in an obligate photoautotroph.     

Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.     

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.     

Hexuronates influence the oligomeric form of the Dps structural protein of bacterial nucleoid and its ability to bind to linear DNA fragments

Proteins of the Dps family perform a dual function in bacterial cells. As ferritins, they protect cells from destructive effects of Fe²⁺ ions, while interacting with DNA they condense the genome in the absence of nutrients. The ability of Dps to self-aggregate is of a great importance. The way of genome remodelling from the condensed state to the active one is not yet known. Here, the effects of two sugar ligands on Dps interaction with DNA have been studied in vitro. For the first time it was demonstrated that D-glucuronate and D-galacturonate, but not D-glucose, can decompose the dodecameric structure of the protein and D-glucuronate stimulated the formation of binary complexes with the linear DNA fragments. As a result of flexible molecular docking, it was found that the molecules of all three sugars potentially can form clusters inside the protein cavity of Dps, but D-glucuronate and D-galacturonate were also bound in the region of intersubunit contacts of oligomer. The consequent destabilization of the intersubunit bonding network can, thus, be the main factor provoking the protein decay to the smaller oligomeric forms. Such a structural rearrangement, leading to a reduction in aggregation, may play a key role in genome decondensation during cell transition to the phase of rapid growth.     

Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps

The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30–60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.