Structure of DPS Protein Complexes with DNA (original) (raw)
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Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy
PLOS ONE, 2015
Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumu-lating~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe 2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.
The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr's salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe 3 O 4 , rather than the expected Fe 2 O 3 , in the sample. Assuming that Fe 2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe 2 O 3 with Dps, but the binding sites with putative functionality were predicted only for Fe 2 O 3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.
The Oligomeric Form of the Escherichia coli Dps Protein Depends on the Availability of Iron Ions
The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr's salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe 3 O 4 , rather than the expected Fe 2 O 3 , in the sample. Assuming that Fe 2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe 2 O 3 with Dps, but the binding sites with putative functionality were predicted only for Fe 2 O 3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.
Nucleic Acids Research, 2004
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, DpsD8 and DpsD18, 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 DpsD8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes. Conversely, DpsD18 does not self-aggregate and binds DNA without causing condensation. At pH 8.2, DpsD8 and DpsD18 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 H 2 O 2 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 Journal of biological chemistry, 2003
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 tur...
International Journal of Molecular Sciences
DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N- and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low- and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectros...
The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner
Dps is a multifunctional homododecameric protein that oxidizes Fe 2+ ions accumulating them in the form of Fe 2 O 3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of
Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations
International Journal of Molecular Sciences
Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cation...
European Biophysics Journal, 2021
DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of twelve equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps-DNA complex formation with an apparent dissociation constant of 5.9 ± 1.0 µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein) DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.
Projection structures reveal the position of the DNA within DNA-Dps Co-crystals
Biochemical and Biophysical Research Communications, 2019
One of the universal mechanisms for the response of Escherichia coli to stress is the increase of the synthesis of specific histone-like proteins that bind the DNA, Dps. As a result, two-and three-dimensional crystalline arrays may be observed in the cytoplasm of starving cells. Here, we determined the conditions to obtain very thin two-dimensional DNA-Dps co-crystals in vitro, and studied their projection structures, using electron microscopy. Analysis of the projection maps of the free Dps crystals revealed two lattice types: hexagonal and rectangular. We used the fluorescently labeled DNA to prove that the DNA is present within the co-crystals with Dps in vitro, and visualized its position using transmission electron microscopy. Molecular modeling confirmed the DNA position within the crystal. We have also suggested a structural model for the DNA-Dps co-crystal dissolving in the presence of Mg 2þ ions.