Dual roles of an essential cysteine residue in activity of a redox-regulated bacterial transcriptional activator.
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.
Gupta
Nirupama
N
Department of Biological Chemistry, University of Michigan, Ann Arbor, Mighigan 48109-0606, USA.
Ragsdale
Stephen W
SW
D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D017930:Genes, Reporter; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004220:Disulfides; D014157:Transcription Factors; D004247:DNA; D003545:Cysteine; D010100:Oxygen