Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR

Abstract

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pKa of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14–Cys49′ intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8–9 Å apart from Cys49′. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4′ helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Methionine oxidation activates a transcription factor in response to oxidative stress

“…Amino acids involved in a defined activity regulation scheme would be expected to be conserved [e.g., the active-site Cys in Hsp33, oxidative stress regulator OxyR, NemR, and HypR (16)(17)(18)33), and the Met in calmodulin kinase II (32)]. However, only M230 and M206 of HypT are located in a highly conserved region; M230 is conserved, whereas M206 is partially conserved and M123 is fairly variable (see alignment in ref.…”

“…The conserved redox-regulated chaperone heat shock protein Hsp33 (16) is activated by Cys oxidation in combination with intrinsic unfolding upon HOCl stress and thus prevents the aggregation of cellular proteins (3). The Bacillus subtilis MarR-type DNA-binding transcriptional repressor hypochloric acid-specific regulator HypR is activated by disulfide-bond formation and confers protection against HOCl via the flavin oxidoreductase HypO (17). The TetR-type regulator Nethylmaleimide reductase repressor (NemR) from Escherichia coli undergoes Cys oxidation upon exposure to HOCl and causes derepression of encoding glyoxalase I (gloA) and encoding Nethylmaleimide reductase (nemA) that were shown to confer HOCl resistance (18).…”

S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress inFirmicutesBacteria

“…(C) Diagonal nonreducing/reducing SDS-PAGE confirmed intermolecular disulfides for AhpC homologs in B. subtilis, B. amyloliquefaciens, B.megaterium, S. carnosus and for YkuU in B. pumilus. The IAM-alkylated proteins extracts (100 lg) of NaOCl-treated cells of all strains were separated by 2D nonreducing/reducing diagonal SDS-PAGE analysis as described previously (41). The AhpC homologs are encircled in the diagonal assays of B. subtilis (AhpC-Bsub), B. amyloliquefaciens (AhpC-Bam), S. carnosus (AhpCScar), B. megaterium (AhpC-Bmeg), and B. pumilus (YkuU-Bpum).…”

The RclR Protein Is a Reactive Chlorine-specific Transcription Factor in Escherichia coli

“…These include the Escherichia coli transcription factors HypT and NemR and the Bacillus subtilis transcription factors OhrR and HypR (18 -21). Interestingly, whereas HypT is so far only known to respond to HOCl (19,22), the other RCS-sensing transcription factors respond to a variety of other stress signals, including cysteinemodifying electrophiles (20,21,23) and organic hydroperoxides (18,24). In contrast to ROS and toxic electrophiles, which have a more limited set of cellular targets (25)(26)(27), RCS are capable of damaging proteins, DNA, lipids, and most other cellular components (1)(2)(3).…”