title: In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes.
abstract: The leucine zipper family of proteins have a DNA binding domain composed of a leucine zipper dimerisation interface and a basic DNA binding structure. We show here that redox changes affect the in vitro DNA binding ability of a select subset of leucine zipper proteins. The bacterially expressed DNA binding domains of Fos/Jun and BZLF1 are unable to bind DNA under non-reducing conditions whereas binding of the C/EBP DNA binding domain is unaffected. Sensitivity to redox state is due to the presence of a conserved cysteine residue in the basic DNA binding motif of Fos, Jun and BZLF1 but not C/EBP. Under non-reducing conditions an intermolecular disulphide bridge is formed between the cysteine residues of each basic motif within a dimer, which prevents DNA binding. We show that oxidation of these C residues can be achieved enzymatically, using glutathione peroxidase, and that DNA binding protects them from oxidation. These data raise the possibility that intracellular changes in the redox state may differentially regulate the activity of leucine zipper family members. In addition the loss of DNA binding activity under non-reducing conditions has implications for the purification methods used to isolate proteins of the leucine zipper family for structural analysis.
authors.affiliation: Department of Pathology, Cambridge University, Addenbrooke's Hospital, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Bannister
authors.forename: A
authors.initials: A
authors.lastname: Cook
authors.forename: T
authors.initials: T
authors.lastname: Kouzarides
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011494:Protein Kinases; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D004247:DNA; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D011494:Protein Kinases; D003545:Cysteine; D004229:Dithiothreitol