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. 2008 Oct 17;283(42):28721-8.
doi: 10.1074/jbc.M800630200. Epub 2008 Aug 7.

Dual roles of an essential cysteine residue in activity of a redox-regulated bacterial transcriptional activator

Nirupama Gupta  1 , Stephen W Ragsdale
Affiliations

Dual roles of an essential cysteine residue in activity of a redox-regulated bacterial transcriptional activator

Nirupama Gupta et al. J Biol Chem. .

Abstract

CprK from Desulfitobacterium dehalogenans is the first characterized transcriptional regulator of anaerobic dehalorespiration and is controlled at two levels: redox and effector binding. In the reduced state and in the presence of chlorinated aromatic compounds, CprK positively regulates expression of the cpr gene cluster. One of the products of the cpr gene cluster is CprA, which catalyzes the reductive dehalogenation of chlorinated aromatic compounds. Redox regulation of CprK occurs through a thiol/disulfide redox switch, which includes two classes of cysteine residues. Under oxidizing conditions, Cys11 and Cys200 form an intermolecular disulfide bond, whereas Cys105 and Cys111 form an intramolecular disulfide. Here, we report that Cys11 is involved in redox inactivation in vivo. Upon replacement of Cys11 with serine, alanine, or aspartate, CprK loses its DNA binding activity. C11A is unstable; however, circular dichroism studies demonstrate that the stability and overall secondary structures of CprK and the C11S and C11D variants are similar. Furthermore, effector binding remains intact in the C11S and C11D variants. However, fluorescence spectroscopic results reveal that the tertiary structures of the C11S and C11D variants differ from that of the wild type protein. Thus, Cys11 plays a dual role as a redox switch and in maintaining the correct tertiary structure that promotes DNA binding.

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Figures

FIGURE 1.
FIGURE 1.
Regulation of CprK. CprK can exist in a thiol-oxidized state, containing an intermolecular disulfide linkage between Cys11 and Cys200 and an intramolecular disulfide linkage between Cys105 and Cys111, or a thiol-reduced state, in which all these Cys residues are in the thiol(ate) form. Oxidized and reduced CprK bind effector (CHPA) with similar affinity. The oxidized state of CprK has low affinity for DNA, whereas the reduced form with effector binds DNA with high affinity and positively regulates expression of the cpr gene cluster. The products of this gene cluster are involved in catalysis of dehalorespiration (10).
FIGURE 2.
FIGURE 2.
In vivo Cys11-Cys200 disulfide bond formation upon treatment of cells with oxidants. Exposure of cells containing wild type CprK to 1 mm DA (A) or 1 mm hydrogen peroxide (B) or after treatment of the C11S variant (C) and the C200S variant (D) with 1 mm DA followed by separation on non reducing SDS-PAGE and Western blot analysis. The membranes were immunoblotted with anti-CprK antibody and stained as described under “Materials and Methods.” WT, wild type.
FIGURE 3.
FIGURE 3.
InvitroDNAbindingactivityofCys11 variants.A, EMSA experiments after incubation of wild type (WT) CprK (lanes 1-4) or C11S (lanes 5-7) with DNA in the presence of DA (lanes 2 and 5), DTT (lanes 3 and 6), or DTT+DA (lanes 4 and 7). B, EMSA experiments comparing wild type CprK with C11D. Lane 2 has wild type CprK with 50 mm DTT. Lanes 3 and 4 have the C11D variant with 50 mm DTT and 5 mm DA, respectively. Lane 1 in A or B is a control that lacks protein.
FIGURE 4.
FIGURE 4.
In vivo DNA binding activity of Cys11 variants. E. coli strains expressing wild type (WT) CprK or Cys11 variants were incubated with no (filled bar) or 1 mm DA (open bar) and promoter activity was measured by the β-galactosidase assay as described under “Materials and Methods.” Activity is expressed as a percentage of Miller units exhibited by the variant compared with that of wild type CprK. The data are shown as the means ± S.D. and are representative of three separate experiments, each performed in quadruplicate.
FIGURE 5.
FIGURE 5.
Effector binding affinity of wild type and Cys11 variants. Fluorescence intensities were measured, as described (10) for the oxidized (right) and reduced (left) wild type (WT, ⋄), C11D (○), and C11S (□) variants. The dashed and solid lines represent the fits to one-site binding models according to Equations 1 and 2, respectively, as described under “Materials and Methods.” Effector concentrations are given in micromolar units. The Kd values are given in Table 1.
FIGURE 6.
FIGURE 6.
Effect of increasing DNA concentration on DNA binding activity of C11S. Wild type (WT) CprK (first three lanes) and the C11S variant (last three lanes) were incubated with 10 nm to 500 nm DNA containing the cpr promoter. The middle lane was a control that lacked protein.
FIGURE 7.
FIGURE 7.
Cys11 in the crystal structure of oxidized CprK. The crystal structure of oxidized D. hafniense CprK (left), focusing on the helix-turn-helix DNA-binding domain (boxed and expanded to the right). Cys11 and other residues predicted to be in the recognition helix, based on the structure of the CRP-DNA complex (10), are shown as space-filling diagrams and colored according to element. This figure was generated from Protein Data Bank code 2h6b in CHIMERA.
FIGURE 8.
FIGURE 8.
Intrinsic fluorescence spectra of CprK. Spectra were recorded for wild type (WT) CprK, C11S and C11D in 50 mm Tris-HCl, 300 mm NaCl, pH 7.5 (A), or the same buffer containing 50 mm DTT (B). Emission spectra were scanned from 300 to 400 nm. The data presented here are representative of three different experiments.
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References

    1. Utkin, I., Woese, C., and Wiegel, J. (1994) Int. J. Syst. Bacteriol. 44 612-619 - PubMed
    1. Villemur, R., Lanthier, M., Beaudet, R., and Lepine, F. (2006) FEMS Microbiol. Rev. 30 706-733 - PubMed
    1. Wiegel, J., Zhang, X., and Wu, Q. (1999) Appl. Environ. Microbiol. 65 2217-2221 - PMC - PubMed
    1. Wiegel, J., and Wu, Q. Z. (2000) FEMS Microbiol. Ecol. 32 1-15 - PubMed
    1. Smidt, H., and de Vos, W. M. (2004) Annu. Rev. Microbiol. 58 43-73 - PubMed

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