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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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Sequence alignment of the HypR-boxes in the hypO and hypR promoter regions. The hypO and hypR promoter sequences (−10 and −35), the transcription start site (+1) and the ATG start codons are underlined in the hypO and hypR upstream regions. The conserved HypR-boxes including the inverted repeats are boxed and indicated by arrows. The HypR protected region identified by the DNase-I footprinting analysis is grey shaded.
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gkr1316-F4: Sequence alignment of the HypR-boxes in the hypO and hypR promoter regions. The hypO and hypR promoter sequences (−10 and −35), the transcription start site (+1) and the ATG start codons are underlined in the hypO and hypR upstream regions. The conserved HypR-boxes including the inverted repeats are boxed and indicated by arrows. The HypR protected region identified by the DNase-I footprinting analysis is grey shaded.

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)

Sequence alignment of the HypR-boxes in the hypO and hypR promoter regions. The hypO and hypR promoter sequences (−10 and −35), the transcription start site (+1) and the ATG start codons are underlined in the hypO and hypR upstream regions. The conserved HypR-boxes including the inverted repeats are boxed and indicated by arrows. The HypR protected region identified by the DNase-I footprinting analysis is grey shaded.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1316-F4: Sequence alignment of the HypR-boxes in the hypO and hypR promoter regions. The hypO and hypR promoter sequences (−10 and −35), the transcription start site (+1) and the ATG start codons are underlined in the hypO and hypR upstream regions. The conserved HypR-boxes including the inverted repeats are boxed and indicated by arrows. The HypR protected region identified by the DNase-I footprinting analysis is grey shaded.
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.

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