Structure of the Dps-Like Protein from Sulfolobus Solfataricus Reveals a Bacterioferritin-Like Dimetal Binding Site within a Dps-Like Dodecameric Assembly.

Oxidative stress, in which reactive oxygen species (ROS1) react indiscriminately with DNA, proteins and lipids, is a universal phenomenon experienced by organisms in all domains of life. Cumulative damage from ROS is now thought to contribute to numerous disease states and a multitude of reports in the primary literature describe the role of oxidative stress in human diseases. Thus, understanding molecular responses to oxidative stress is of significant medical importance. Although much work has been done to characterize the management of oxidative stress in eukaryotes and bacteria, the oxidative stress response in archaea is poorly understood. However, elucidation of the protective mechanisms utilized in archaea is certain to provide insight into the diversity of molecular responses to oxidative stress across all domains of life. Numerous mechanisms have evolved to minimize and repair the damaging effects of ROS. These include enzymes such as superoxide dismutase and superoxide reductase, which convert the superoxide ion into molecular oxygen (O2) and/or hydrogen peroxide (H2O2). Hydrogen peroxide is generated in other ways as well and is, in fact, a natural product of the cell. Although hydrogen peroxide is a relatively moderate oxidant, its interaction with Fe2+ through the Fenton reaction results in the production of the more destructive hydroxyl radical (eq I). The combination of Fe2+ and H2O2 thus represents a lethal combination for life, and mechanisms for the elimination of H2O2 are essential. H2O2+Fe2+→Fe3++OH-+HO· (I) Catalase represents one means per destruction of unwanted H2O2, where disproportionation of H2O2 results in the production of water and molecular oxygen. An alternative strategy is the use of various peroxidase activities, including glutathione and thioredoxin dependent peroxidases, to reduce H2O2 to H2O while avoiding the production of molecular oxygen. A third strategy for minimizing hydroxyl radical production by the Fenton reaction is to minimize the concentration of free ferrous ion. This strategy is employed in the mineralization reactions catalyzed by ferritin, bacterioferritin, and DPS proteins (DNA binding protein from starved cells), all members of the ferritin superfamily (1–3). In the case of ferritin and bacterioferritin, ferrous ion and oxygen react at the ferroxidase center, eventually leading to the deposition of a benign insoluble ferric oxide core within the hollow, spherically shaped protein particles. DPS catalyzes a similar reaction. However, in contrast to ferritin, DPS clearly prefers H2O2 as the oxidant (4, 5). Thus, through reaction with H2O2, the activity of DPS simultaneously mitigates the toxicity of H2O2 through a 2-electron reduction to form water, while at the same time, it inactivates the Fe2+ by oxidation to Fe3+. Recent work indicates that bacterioferritin can also utilize H2O2 as an oxidant (5, 6); hence, bacterioferritin will also catalyze the simultaneous consumption of both Fenton reactants. The secondary and tertiary structures in DPS molecules are largely homologous to those of ferritin and bacterioferritin (7). The structural core of these proteins is a highly conserved four-helix bundle, composed of two consecutive helix-turn-helix motifs. The two helix-turn-helix motifs are related to each other by pseudo 2-fold symmetry and are connected to each other by an extended loop that runs the length of the four helix bundle. In structurally characterized DPS proteins, a fifth helix is found within this loop, where it helps to define the 2-fold symmetric subunit interface (7–9). Although DPS, ferritin, and bacterioferritin all serve to protect the organism from oxidative damage by sequestering intracellular ferrous iron, the active site at which iron is bound and oxidized in DPS is different from that of ferritin and bacterioferritin. In ferritin and bacterioferritin, iron is found in a di-iron carboxylate binding motif located within the four-helix bundle. In contrast, the structurally characterized DPS proteins show an iron binding site located at the interface between two subunits related by 2-fold symmetry (5, 10, 11). A second difference between bacterioferritin and DPS proteins is the nature of their oligomeric assemblies. While ferritin and bacterioferritin are typically found as 24-mers, with 432 point group symmetry, DPS is found as a dodecamer (12-mer), with 23 point group symmetry (7). We have previously reported the isolation and characterization of DPS-like (DPSL) proteins from Sulfolobus solfataricus (SsDPSL) (12) and Pyrococcus furiosus (13) and described their role as antioxidant proteins in vitro. On the basis of phylogenetic analysis, these proteins, along with eight others, were shown to form a monophyletic cluster that is distinct from all other protein subclasses within the ferritin superfamily. Biochemical characterization of these DPSL proteins reveals dodecameric assemblies that preferentially utilize H2O2 in the controlled oxidation of Fe2+ (12), and an N-terminal extension similar to that present in DPS molecules, which by analogy, is thought to mediate an interaction with DNA. On the basis of the observed antioxidant properties, oligomeric state, and N-terminal extension of the prototypical proteins from Pyrococcus furiosus and Sulfolobus solfataricus, this subclass of proteins was termed DPS-like (12). To date, the nature of the ferroxidase center in these DPSL proteins has been less clear. Analogous to authentic DPS proteins, candidate residues for an intersubunit di-iron binding site in the DPSL proteins have been suggested (12). However, residues corresponding to a bacterioferritin-like ferroxidase center are also found within the sequences of all DPSL proteins (13). Thus, it is not surprising that DPSL proteins are classified within Pfam (14) as a bacterioferritin-like subfamily (CD 1052.2) that lacks residues involved in heme binding. Given the extensive biochemical similarities between DPS and DPSL proteins, the functional properties responsible for preservation of the DPSL signature across a diverse group of prokaryotes are unclear. So too the nature of the ferroxidase site itself. In an effort to illuminate the basis for this distinct phylogenetic signature, we have undertaken structural studies of DPSL proteins. Here, we describe the structure of SsDPSL, the DPS-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus.