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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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HypR is oxidized to Cys14–Cys49′ intersubunit disulphides in response to diamide and NaOCl in vitro (A and B) and in vivo (C and D). (A) His-HypR protein was treated with 1 mM diamide or 100 µM NaOCl, alkylated with 50 mM IAM and subjected to non-reducing SDS–PAGE analysis. The bands corresponding to the oxidized HypR disulphide dimer were tryptically digested and analysed using LTQ-Orbitrap mass spectrometry. (B) The Cys14–Cys49'-intermolecular disulphide-containing peptide was observed as quadruply charged precursor ion at an m/z = 620.3151. The CID MS/MS spectrum of this Cys14–Cys49′-disulphide peptide is shown with b and y ion fragment ions in red and blue. The detailed MS/MS data for this peptide including the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and the fragment ion seria are presented in Supplementary Figure S6. (C) 1D-western blot analysis and (D) 2D-diagonal western blot analysis of immunoprecipitated HypR protein purified from wild-type cells before (co) and after exposure to diamide and NaOCl in the presence of 50 mM IAM using HypR-specific polyclonal antibodies. The HypR-specific intersubunit disulphide is indicated by an arrow that was also visible in non-reducing SDS-gels and analysed using Orbitrap LC–MS/MS. The CID MS/MS spectrum, the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and fragment ion seria of this Cys14–Cys49′-disulphide peptide of HypR purified from cells in vivo are presented in Supplementary Figure S6.
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gkr1316-F6: HypR is oxidized to Cys14–Cys49′ intersubunit disulphides in response to diamide and NaOCl in vitro (A and B) and in vivo (C and D). (A) His-HypR protein was treated with 1 mM diamide or 100 µM NaOCl, alkylated with 50 mM IAM and subjected to non-reducing SDS–PAGE analysis. The bands corresponding to the oxidized HypR disulphide dimer were tryptically digested and analysed using LTQ-Orbitrap mass spectrometry. (B) The Cys14–Cys49'-intermolecular disulphide-containing peptide was observed as quadruply charged precursor ion at an m/z = 620.3151. The CID MS/MS spectrum of this Cys14–Cys49′-disulphide peptide is shown with b and y ion fragment ions in red and blue. The detailed MS/MS data for this peptide including the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and the fragment ion seria are presented in Supplementary Figure S6. (C) 1D-western blot analysis and (D) 2D-diagonal western blot analysis of immunoprecipitated HypR protein purified from wild-type cells before (co) and after exposure to diamide and NaOCl in the presence of 50 mM IAM using HypR-specific polyclonal antibodies. The HypR-specific intersubunit disulphide is indicated by an arrow that was also visible in non-reducing SDS-gels and analysed using Orbitrap LC–MS/MS. The CID MS/MS spectrum, the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and fragment ion seria of this Cys14–Cys49′-disulphide peptide of HypR purified from cells in vivo are presented in Supplementary Figure S6.

Mentions: To analyse the oxidative modification of HypR in vitro, purified HypR-His-protein was treated with diamide and NaOCl and separated using non-reducing SDS–PAGE. HypR is reversibly oxidized to intersubunit disulphides by diamide and NaOCl stress since it migrates at the size of the HypR dimer upon oxidation (Figure 6A). In contrast, the majority of HypRC14S and HypRC49S mutant proteins do not form intermolecular disulphides upon oxidation.Figure 6.


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)

HypR is oxidized to Cys14–Cys49′ intersubunit disulphides in response to diamide and NaOCl in vitro (A and B) and in vivo (C and D). (A) His-HypR protein was treated with 1 mM diamide or 100 µM NaOCl, alkylated with 50 mM IAM and subjected to non-reducing SDS–PAGE analysis. The bands corresponding to the oxidized HypR disulphide dimer were tryptically digested and analysed using LTQ-Orbitrap mass spectrometry. (B) The Cys14–Cys49'-intermolecular disulphide-containing peptide was observed as quadruply charged precursor ion at an m/z = 620.3151. The CID MS/MS spectrum of this Cys14–Cys49′-disulphide peptide is shown with b and y ion fragment ions in red and blue. The detailed MS/MS data for this peptide including the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and the fragment ion seria are presented in Supplementary Figure S6. (C) 1D-western blot analysis and (D) 2D-diagonal western blot analysis of immunoprecipitated HypR protein purified from wild-type cells before (co) and after exposure to diamide and NaOCl in the presence of 50 mM IAM using HypR-specific polyclonal antibodies. The HypR-specific intersubunit disulphide is indicated by an arrow that was also visible in non-reducing SDS-gels and analysed using Orbitrap LC–MS/MS. The CID MS/MS spectrum, the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and fragment ion seria of this Cys14–Cys49′-disulphide peptide of HypR purified from cells in vivo are presented in Supplementary Figure S6.
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gkr1316-F6: HypR is oxidized to Cys14–Cys49′ intersubunit disulphides in response to diamide and NaOCl in vitro (A and B) and in vivo (C and D). (A) His-HypR protein was treated with 1 mM diamide or 100 µM NaOCl, alkylated with 50 mM IAM and subjected to non-reducing SDS–PAGE analysis. The bands corresponding to the oxidized HypR disulphide dimer were tryptically digested and analysed using LTQ-Orbitrap mass spectrometry. (B) The Cys14–Cys49'-intermolecular disulphide-containing peptide was observed as quadruply charged precursor ion at an m/z = 620.3151. The CID MS/MS spectrum of this Cys14–Cys49′-disulphide peptide is shown with b and y ion fragment ions in red and blue. The detailed MS/MS data for this peptide including the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and the fragment ion seria are presented in Supplementary Figure S6. (C) 1D-western blot analysis and (D) 2D-diagonal western blot analysis of immunoprecipitated HypR protein purified from wild-type cells before (co) and after exposure to diamide and NaOCl in the presence of 50 mM IAM using HypR-specific polyclonal antibodies. The HypR-specific intersubunit disulphide is indicated by an arrow that was also visible in non-reducing SDS-gels and analysed using Orbitrap LC–MS/MS. The CID MS/MS spectrum, the Xcorr, ΔCn scores, precursor ion and neutral molecular masses of the peptide and fragment ion seria of this Cys14–Cys49′-disulphide peptide of HypR purified from cells in vivo are presented in Supplementary Figure S6.
Mentions: To analyse the oxidative modification of HypR in vitro, purified HypR-His-protein was treated with diamide and NaOCl and separated using non-reducing SDS–PAGE. HypR is reversibly oxidized to intersubunit disulphides by diamide and NaOCl stress since it migrates at the size of the HypR dimer upon oxidation (Figure 6A). In contrast, the majority of HypRC14S and HypRC49S mutant proteins do not form intermolecular disulphides upon oxidation.Figure 6.

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.

Show MeSH
Related in: MedlinePlus