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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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The pH dependence of the reactivity of Cys14 and Cys49 with DTNB. The absorbance at 410 nm at different pH values from 6 to 8 resulting from the release of 2-nitro-5 thiobenzoate after reaction of Cys49 of the HypRC14S (A) and of Cys14 of the HypRC49S (B) mutant proteins with DTNB is plotted over the time. (C and D) The kinetic constants (k) of the Cys49 and Cys14 reactions with DTNB are plotted against the pH and fitted resulting in a pKa of 8.51 for Cys49 (C) and a pKa of 6.36 for Cys14 (D).
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gkr1316-F8: The pH dependence of the reactivity of Cys14 and Cys49 with DTNB. The absorbance at 410 nm at different pH values from 6 to 8 resulting from the release of 2-nitro-5 thiobenzoate after reaction of Cys49 of the HypRC14S (A) and of Cys14 of the HypRC49S (B) mutant proteins with DTNB is plotted over the time. (C and D) The kinetic constants (k) of the Cys49 and Cys14 reactions with DTNB are plotted against the pH and fitted resulting in a pKa of 8.51 for Cys49 (C) and a pKa of 6.36 for Cys14 (D).

Mentions: We used the reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to analyse the reactivity of both cysteines at different pH values using His-tagged HypRC14S and His-HypRC49S mutant proteins (Figure 8). Spectroscopic measurement of the released 2-nitro-5 thiobenzoate allows determination of the fraction of deprotonated thiol groups and thus the pKa value. The Cys14 thiol has a more acidic pKa of 6.36 ± 0.04 (random error calculated from the second fit) suggesting that Cys14 is present as thiolate anion and a preferred target for oxidation by diamide or NaOCl (Figure 8B). In contrast, the pKa of Cys49 was determined as 8.51 ± 0.07 indicating that Cys49 is present in its protonated thiol form at physiological pH values in the cytoplasm (Figure 8A). The observed pKa value of the thiol group of Cys49 is also in accordance to its localization in a hydrophobic environment provided by Ile52, Trp27, Ile30, Leu31, Gln60Cγ and Ile48. The sulphur atoms of Cys14 (presumed to be at the position of Ser14Oγ) and Cys49′ residues of the opposing subunits are ∼8–9 Å apart in the HypR dimer (9.0, 8.5, 8.0 and 8.3 Å in monomers A, B, C and D, respectively).Figure 8.


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)

The pH dependence of the reactivity of Cys14 and Cys49 with DTNB. The absorbance at 410 nm at different pH values from 6 to 8 resulting from the release of 2-nitro-5 thiobenzoate after reaction of Cys49 of the HypRC14S (A) and of Cys14 of the HypRC49S (B) mutant proteins with DTNB is plotted over the time. (C and D) The kinetic constants (k) of the Cys49 and Cys14 reactions with DTNB are plotted against the pH and fitted resulting in a pKa of 8.51 for Cys49 (C) and a pKa of 6.36 for Cys14 (D).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351151&req=5

gkr1316-F8: The pH dependence of the reactivity of Cys14 and Cys49 with DTNB. The absorbance at 410 nm at different pH values from 6 to 8 resulting from the release of 2-nitro-5 thiobenzoate after reaction of Cys49 of the HypRC14S (A) and of Cys14 of the HypRC49S (B) mutant proteins with DTNB is plotted over the time. (C and D) The kinetic constants (k) of the Cys49 and Cys14 reactions with DTNB are plotted against the pH and fitted resulting in a pKa of 8.51 for Cys49 (C) and a pKa of 6.36 for Cys14 (D).
Mentions: We used the reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to analyse the reactivity of both cysteines at different pH values using His-tagged HypRC14S and His-HypRC49S mutant proteins (Figure 8). Spectroscopic measurement of the released 2-nitro-5 thiobenzoate allows determination of the fraction of deprotonated thiol groups and thus the pKa value. The Cys14 thiol has a more acidic pKa of 6.36 ± 0.04 (random error calculated from the second fit) suggesting that Cys14 is present as thiolate anion and a preferred target for oxidation by diamide or NaOCl (Figure 8B). In contrast, the pKa of Cys49 was determined as 8.51 ± 0.07 indicating that Cys49 is present in its protonated thiol form at physiological pH values in the cytoplasm (Figure 8A). The observed pKa value of the thiol group of Cys49 is also in accordance to its localization in a hydrophobic environment provided by Ile52, Trp27, Ile30, Leu31, Gln60Cγ and Ile48. The sulphur atoms of Cys14 (presumed to be at the position of Ser14Oγ) and Cys49′ residues of the opposing subunits are ∼8–9 Å apart in the HypR dimer (9.0, 8.5, 8.0 and 8.3 Å in monomers A, B, C and D, respectively).Figure 8.

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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Related in: MedlinePlus