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

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.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'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

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 pK(a) 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.

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In vitro transcription analysis of hypO in the presence of RNA polymerase holoenzyme (RNAP) and purified HypR (A and B) and HypRC14S proteins (C and D) treated with DTT, diamide or NaOCl. The reactions in (A) and (C) show increased hypO transcription ratios by oxidized HypR but not by oxidized HypRC14S mutant protein. The reactions in (B) and (D) show reduced hypO transcription ratios by oxidized HypR that is subsequently reduced with DTT, but no change in hypO transcription by oxidized HypRC14S that is subsequently reduced with DTT. The in vitro-transcription analyses of hypO are representives of three replicate experiments and the relative transcription ratios were quantified in Supplementary Figure S4C. RNA size standard was generated using the Perfect RNA marker template mix (Novagen). The hypO specific run-off transcript is labelled at a size of 220 bp.
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gkr1316-F5: In vitro transcription analysis of hypO in the presence of RNA polymerase holoenzyme (RNAP) and purified HypR (A and B) and HypRC14S proteins (C and D) treated with DTT, diamide or NaOCl. The reactions in (A) and (C) show increased hypO transcription ratios by oxidized HypR but not by oxidized HypRC14S mutant protein. The reactions in (B) and (D) show reduced hypO transcription ratios by oxidized HypR that is subsequently reduced with DTT, but no change in hypO transcription by oxidized HypRC14S that is subsequently reduced with DTT. The in vitro-transcription analyses of hypO are representives of three replicate experiments and the relative transcription ratios were quantified in Supplementary Figure S4C. RNA size standard was generated using the Perfect RNA marker template mix (Novagen). The hypO specific run-off transcript is labelled at a size of 220 bp.

Mentions: Next, we analysed whether transcription of hypO is activated by oxidized HypR using an in vitro transcription assay. In brief, using E. coli RNA polymerase core enzyme (RNAP) and purified SigmaA and HypR proteins from B. subtilis we performed the in vitro transcription assay for the hypO gene as described previously (33). This assay showed the production of a hypO run-off transcript of the expected size of 220 bp only when HypR was present in the in vitro reactions, indicating that HypR activates transcription of the hypO gene in vitro (Figure 5). Transcription of hypO increased ∼2.5-fold when HypR protein was oxidized by diamide and NaOCl treatment compared to reactions with DTT-reduced HypR (Figure 5A and Supplementary Figure S4C). This indicates that the increased DNA-binding affinity of oxidized HypR protein to the hypO promoter observed in the DNase-I footprinting analyses, results in increased activation of hypO transcription in vitro. In addition, hypO specific transcription ratios decreased in the in vitro transcription reactions containing oxidized HypR protein that was subsequently reduced with DTT (Figure 5B and Supplementary Figure S4C). In contrast, transcription of hypO was not increased by oxidation of HypRC14S mutant protein in vitro and also not affected by oxidized HypRC14S protein that was treated subsequently with DTT (Figure 5C and D; Supplementary Figure S4C). These results show that oxidation of HypR increases activation of hypO transcription by the RNAP in vitro.Figure 5.


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

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

In vitro transcription analysis of hypO in the presence of RNA polymerase holoenzyme (RNAP) and purified HypR (A and B) and HypRC14S proteins (C and D) treated with DTT, diamide or NaOCl. The reactions in (A) and (C) show increased hypO transcription ratios by oxidized HypR but not by oxidized HypRC14S mutant protein. The reactions in (B) and (D) show reduced hypO transcription ratios by oxidized HypR that is subsequently reduced with DTT, but no change in hypO transcription by oxidized HypRC14S that is subsequently reduced with DTT. The in vitro-transcription analyses of hypO are representives of three replicate experiments and the relative transcription ratios were quantified in Supplementary Figure S4C. RNA size standard was generated using the Perfect RNA marker template mix (Novagen). The hypO specific run-off transcript is labelled at a size of 220 bp.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr1316-F5: In vitro transcription analysis of hypO in the presence of RNA polymerase holoenzyme (RNAP) and purified HypR (A and B) and HypRC14S proteins (C and D) treated with DTT, diamide or NaOCl. The reactions in (A) and (C) show increased hypO transcription ratios by oxidized HypR but not by oxidized HypRC14S mutant protein. The reactions in (B) and (D) show reduced hypO transcription ratios by oxidized HypR that is subsequently reduced with DTT, but no change in hypO transcription by oxidized HypRC14S that is subsequently reduced with DTT. The in vitro-transcription analyses of hypO are representives of three replicate experiments and the relative transcription ratios were quantified in Supplementary Figure S4C. RNA size standard was generated using the Perfect RNA marker template mix (Novagen). The hypO specific run-off transcript is labelled at a size of 220 bp.
Mentions: Next, we analysed whether transcription of hypO is activated by oxidized HypR using an in vitro transcription assay. In brief, using E. coli RNA polymerase core enzyme (RNAP) and purified SigmaA and HypR proteins from B. subtilis we performed the in vitro transcription assay for the hypO gene as described previously (33). This assay showed the production of a hypO run-off transcript of the expected size of 220 bp only when HypR was present in the in vitro reactions, indicating that HypR activates transcription of the hypO gene in vitro (Figure 5). Transcription of hypO increased ∼2.5-fold when HypR protein was oxidized by diamide and NaOCl treatment compared to reactions with DTT-reduced HypR (Figure 5A and Supplementary Figure S4C). This indicates that the increased DNA-binding affinity of oxidized HypR protein to the hypO promoter observed in the DNase-I footprinting analyses, results in increased activation of hypO transcription in vitro. In addition, hypO specific transcription ratios decreased in the in vitro transcription reactions containing oxidized HypR protein that was subsequently reduced with DTT (Figure 5B and Supplementary Figure S4C). In contrast, transcription of hypO was not increased by oxidation of HypRC14S mutant protein in vitro and also not affected by oxidized HypRC14S protein that was treated subsequently with DTT (Figure 5C and D; Supplementary Figure S4C). These results show that oxidation of HypR increases activation of hypO transcription by the RNAP in vitro.Figure 5.

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.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'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

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 pK(a) 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.

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