Transcriptional activation of dehalorespiration. Identification of redox-active cysteines regulating dimerization and DNA binding.
Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.
Pop
Stelian M
SM
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Gupta
Nirupama
N
Raza
Ashraf S
AS
Ragsdale
Stephen W
SW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045858:Desulfitobacterium / Q000235:genetics / Q000378:metabolism; D003958:Diamide / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011078:Polychlorinated Biphenyls / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004220:Disulfides; D007506:Iron-Sulfur Proteins; D010455:Peptides; D021122:Protein Subunits; D013439:Sulfhydryl Reagents; D014157:Transcription Factors; D003958:Diamide; D011078:Polychlorinated Biphenyls; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol