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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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DNase-I footprinting experiments (A) and gel-shift experiments (B) of purified HypR protein to the hypO promoter in the presence of DTT, diamide and NaOCl. (A) The HypR-protected operator sequence is indicated at the right side of the DNase-I footprint including an 7-2-7 bp inverted repeat with the sequence GTATCAAAATTGATAC that is labelled by arrows in the sequence alignment in Figure 4. The positions relative to the transcriptional start site are shown on the left. Transcription start site is indicated by (+1). For dideoxynucleotide sequencing, the dideoxy nucleotide added in each reaction is indicated above the corresponding lane. HypR protein was treated with 1 mM DTT or 1 mM diamide prior to the DNA-binding reactions. (B) EMSAs were used to analyse the effect of DTT, 1 mM diamide and 100 µM NaOCl on the DNA-binding activity of purified HypR, HypRC14S and HypRC49S proteins to the labelled hypO promoter probe. The HypR protein amounts used for the DNA-binding reactions are indicated. The EMSA experiments are representives of three replicate experiments. The change in the DNA-binding affinity and dissociation constants (Kd) of reduced and oxidized HypR, HypRC14S and HypRC49S proteins are calculated in Supplementary Figure S4A and B.
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gkr1316-F3: DNase-I footprinting experiments (A) and gel-shift experiments (B) of purified HypR protein to the hypO promoter in the presence of DTT, diamide and NaOCl. (A) The HypR-protected operator sequence is indicated at the right side of the DNase-I footprint including an 7-2-7 bp inverted repeat with the sequence GTATCAAAATTGATAC that is labelled by arrows in the sequence alignment in Figure 4. The positions relative to the transcriptional start site are shown on the left. Transcription start site is indicated by (+1). For dideoxynucleotide sequencing, the dideoxy nucleotide added in each reaction is indicated above the corresponding lane. HypR protein was treated with 1 mM DTT or 1 mM diamide prior to the DNA-binding reactions. (B) EMSAs were used to analyse the effect of DTT, 1 mM diamide and 100 µM NaOCl on the DNA-binding activity of purified HypR, HypRC14S and HypRC49S proteins to the labelled hypO promoter probe. The HypR protein amounts used for the DNA-binding reactions are indicated. The EMSA experiments are representives of three replicate experiments. The change in the DNA-binding affinity and dissociation constants (Kd) of reduced and oxidized HypR, HypRC14S and HypRC49S proteins are calculated in Supplementary Figure S4A and B.

Mentions: DNase-I footprinting analysis was performed to identify the cis-acting sequences which function as operator sites for HypR binding in the hypO upstream region in vitro. HypR-His protein protected a region upstream of the hypO promoter from positions −53 to −95 relative to the transcription start site (Figure 3A). The protected region contains a 7-2-7 bp inverted repeat GTATCAAAATTGATAC that is also present at positions +24 to +39 downstream of the hypR promoter (Figure 4). The position of this HypO-box confirms the notion that HypR is a positive transcriptional regulator of hypO transcription, but probably represses its own transcription. Furthermore, we were interested if the DNA-binding activity is affected by diamide and NaOCl in vitro and performed gel-shift and DNase-I footprinting analysis of HypR protein under reduced and oxidized conditions. The gel-shift experiments showed binding of HypR to the hypO operator sites at similar affinities under reduced and oxidized conditions (Figure 3B). The calculated dissociation constants (Kd) were 0.18 µM for reduced HypR, 0.14 µM for diamide-oxidized HypR and 0.12 µM for NaOCl-oxidized HypR proteins (Supplementary Figure S4A and S4B). This indicates no significant change in the DNA-binding affinities of reduced and oxidized HypR proteins. Similar Kd values were calculated for reduced HypRC14S and HypRC49S mutant proteins with 0.14 and 0.12 µM, respectively and oxidation caused no significant change in the DNA-binding affinities of the Cys mutant proteins (Supplementary Figure S4A and S4B). However, we observed a change in the mobility of oxidized HypR compared to reduced HypR in the gel-shift assays which was DTT-reversible (Figure 3B). In addition, the DNase-I footprinting analysis showed a higher affinity of oxidized HypR protein to the hypO promoter region indicating an increased DNA-binding activity of oxidized HypR protein in vitro (Figure 3A).Figure 3.


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)

DNase-I footprinting experiments (A) and gel-shift experiments (B) of purified HypR protein to the hypO promoter in the presence of DTT, diamide and NaOCl. (A) The HypR-protected operator sequence is indicated at the right side of the DNase-I footprint including an 7-2-7 bp inverted repeat with the sequence GTATCAAAATTGATAC that is labelled by arrows in the sequence alignment in Figure 4. The positions relative to the transcriptional start site are shown on the left. Transcription start site is indicated by (+1). For dideoxynucleotide sequencing, the dideoxy nucleotide added in each reaction is indicated above the corresponding lane. HypR protein was treated with 1 mM DTT or 1 mM diamide prior to the DNA-binding reactions. (B) EMSAs were used to analyse the effect of DTT, 1 mM diamide and 100 µM NaOCl on the DNA-binding activity of purified HypR, HypRC14S and HypRC49S proteins to the labelled hypO promoter probe. The HypR protein amounts used for the DNA-binding reactions are indicated. The EMSA experiments are representives of three replicate experiments. The change in the DNA-binding affinity and dissociation constants (Kd) of reduced and oxidized HypR, HypRC14S and HypRC49S proteins are calculated in Supplementary Figure S4A and B.
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gkr1316-F3: DNase-I footprinting experiments (A) and gel-shift experiments (B) of purified HypR protein to the hypO promoter in the presence of DTT, diamide and NaOCl. (A) The HypR-protected operator sequence is indicated at the right side of the DNase-I footprint including an 7-2-7 bp inverted repeat with the sequence GTATCAAAATTGATAC that is labelled by arrows in the sequence alignment in Figure 4. The positions relative to the transcriptional start site are shown on the left. Transcription start site is indicated by (+1). For dideoxynucleotide sequencing, the dideoxy nucleotide added in each reaction is indicated above the corresponding lane. HypR protein was treated with 1 mM DTT or 1 mM diamide prior to the DNA-binding reactions. (B) EMSAs were used to analyse the effect of DTT, 1 mM diamide and 100 µM NaOCl on the DNA-binding activity of purified HypR, HypRC14S and HypRC49S proteins to the labelled hypO promoter probe. The HypR protein amounts used for the DNA-binding reactions are indicated. The EMSA experiments are representives of three replicate experiments. The change in the DNA-binding affinity and dissociation constants (Kd) of reduced and oxidized HypR, HypRC14S and HypRC49S proteins are calculated in Supplementary Figure S4A and B.
Mentions: DNase-I footprinting analysis was performed to identify the cis-acting sequences which function as operator sites for HypR binding in the hypO upstream region in vitro. HypR-His protein protected a region upstream of the hypO promoter from positions −53 to −95 relative to the transcription start site (Figure 3A). The protected region contains a 7-2-7 bp inverted repeat GTATCAAAATTGATAC that is also present at positions +24 to +39 downstream of the hypR promoter (Figure 4). The position of this HypO-box confirms the notion that HypR is a positive transcriptional regulator of hypO transcription, but probably represses its own transcription. Furthermore, we were interested if the DNA-binding activity is affected by diamide and NaOCl in vitro and performed gel-shift and DNase-I footprinting analysis of HypR protein under reduced and oxidized conditions. The gel-shift experiments showed binding of HypR to the hypO operator sites at similar affinities under reduced and oxidized conditions (Figure 3B). The calculated dissociation constants (Kd) were 0.18 µM for reduced HypR, 0.14 µM for diamide-oxidized HypR and 0.12 µM for NaOCl-oxidized HypR proteins (Supplementary Figure S4A and S4B). This indicates no significant change in the DNA-binding affinities of reduced and oxidized HypR proteins. Similar Kd values were calculated for reduced HypRC14S and HypRC49S mutant proteins with 0.14 and 0.12 µM, respectively and oxidation caused no significant change in the DNA-binding affinities of the Cys mutant proteins (Supplementary Figure S4A and S4B). However, we observed a change in the mobility of oxidized HypR compared to reduced HypR in the gel-shift assays which was DTT-reversible (Figure 3B). In addition, the DNase-I footprinting analysis showed a higher affinity of oxidized HypR protein to the hypO promoter region indicating an increased DNA-binding activity of oxidized HypR protein in vitro (Figure 3A).Figure 3.

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