ST1710–DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators

Thirumananseri Kumarevel Tomoyuki Tanaka Takashi Umehara Shigeyuki Yokoyama ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 A resolutions, respectively. The ST1710-DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism. - Microbial antibiotic resistance is a result of either inactivation or reduced accumulation of antibiotics within an organism. Proteins belonging to the multiple antibiotic resistance regulators (MarR) family reportedly regulate the expression of proteins conferring resistance to multiple antibiotics, organic solvents, household disinfectants, oxidative stress agents and pathogenic factors (13). For example, in the absence of the appropriate stimulus, Escherichia coli MarR proteins negatively regulate the marRAB operon, and repression of this operon is alleviated by exposure to a variety of phenolic compounds, most notably sodium salicylate (1). Similarly, MexR negatively regulates an operon in Pseudomonas aeruginosa that, when expressed, encodes a tri-partite multi-drug efflux system that results in an increased resistance to multiple antibiotics, including tetracycline, b-lactams, chloramphenicol, novobiocin, trimethoprim, sulfonamides and fluoroquinolones (4,5). Some members of the MarR family of DNA-binding proteins, for example hypothetical uricase regulator (HucR) and organic hydroperoxide resistance regulator (OhrR), mediate a cellular response to reactive oxidative stress (ROS) (6,7). The Deinococcus radiodurans HucR was shown to repress its own expression as well as that of an uricase. This repression is alleviated both in vivo and in vitro upon binding uric acid, the substrate for uricase. As uric acid is a potent scavenger of reactive oxygen species, and D. radiodurans is known for its remarkable resistance to DNA-damaging agents, these observations indicate a novel oxidative stress response mechanism (810). Similar to HucR, the OhrR protein of Bacillus subtilis also mediates a response to oxidative stress; however, for OhrR, it is oxidation of a lone cysteine residue by organic hydroperoxides that abrogates DNA binding (11,12). We have reported two different crystal forms of ST1710 (13) and others (14). The structure showed the winged helix-turn-helix (wHtH) motif at the DNA binding site that obviously belonged to the MarR family of proteins. The crystal structures of proteins in the MarR family have also been determined from a number of organisms including MarR from E. coli (15), MexR from P. aeruginosa (16), SarR from Staphylococcus aureus (17), Slya-like protein from Enterococcus faecalis (18), OhrR from B. subtilis (19), HucR from D. radiodurans (20) and MTH313 from Methanobacterium thermoautotrophicum (21). Sequence comparisons of these proteins with ST1710 showed less than 25% identity. A homology search in the non-redundant protein database using Blastp revealed that ST1710 has about 51% identity to the Sulfolobus acidocaldarius (22) and Sulfolobus solfataricus (23) sequences and about 41% identity to the Metallosphaera sedula sequence (24). However, none of the proteins closely related to ST1710 have been biochemically or structurally characterized. Sodium salicylate is well known to inhibit MarR activity both in vitro and in vivo, at millimolar concentration levels (25). Sodium salicylate is routinely used as a model inhibitor of MarR to induce MarA expression in E. coli and Salmonella typhimurium, thereby conferring a Mar phenotype (2528). The structure of E. coli MarR was solved with two salicylate molecules per monomer and both of them are highly exposed to the solvent. It seems that salicylate may have stabilized the crystal packing since in the absence of salicylate, the crystals could not be used for structure determination in the case of E. coli MarR (15). Recently, the structure of MTH313, a MarR homolog from M. thermoautotrophicum, was solved in the free form and complexed with salicylate; these analyses revealed a large asymmetrical conformational change that is mediated by the binding of sodium salicylate to two distinct locations in the dimer (21). The members of the MarR family of regulatory proteins recognize double stranded DNA by their wHtH motifs (1519). Footprinting experiments revealed that MarR binds as a dimer at two different, but similar, sites in marO, protecting 21 bp of DNA on both strands at a single site without bending its target (29,30). One of the MarR families of proteins, the OhrR protein complexed with the ohrA operator with a 29-bp duplex was solved, which revealed the interactions between them. The proteinDNA contact region included the major groove of the 10 element, and indicated that OhrR, and probably MarR and MexR as well, repress transcription by blocking the access of RNA polymerase to this promoter element (19). In addition, the mutational analysis of the RNA polymerase binding site, the 10 element of the OhrR-, MarR- and MexR-regulated promoters, revealed the loss of DNA binding ability by 10-fold when this region was altered (11,16,30,31). On the basis of the sequence of the ohrA promoter, we previously identified a putative promoter for ST1710 and showed binding ability by gel-mobility shift assays (13). This promoter is located immediately upstream of the first ATG of the ST1710 gene and downstream of the STS1709 gene. To understand the importance of MarR family members in antibiotic resistance and other biological processes, here, we solved the ST1710 in three different forms: (i) apo-form (native), (ii) complexed with its inhibitor, sodium salicylate (salicylate complex) and (iii) complexed with its promoter DNA. A slight conformational change on the side chains of protein residues was observed when bound to the salicylate ligand, compared to the apo-form. The DNA bound to the wHtH motif of one monomer on the dimeric ST1710, and specifically interacted with the residues R84, R89, R90 and K91. A significantly large conformational change was observed between the monomers of the dimeric protein bound to the DNA, and also with the apo/salicylate complex structures. Significantly, a distinct mode of DNA binding was observed with the bound DNA pa (...truncated)