title: DNA-binding activity of the ERp57 C-terminal domain is related to a redox-dependent conformational change.
abstract: ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a' domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a' domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a' domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a' domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.
authors.affiliation: Department of Biochemical Sciences A. Rossi Fanelli, CNR Institute of Molecular Biology and Pathology and Istituto Pasteur-Fondazione Cenci Bolognetti, University La Sapienza, 00185 Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Grillo
authors.forename: Chiara
authors.initials: C
authors.lastname: D'Ambrosio
authors.forename: Valerio
authors.initials: V
authors.lastname: Consalvi
authors.forename: Roberta
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Manola
authors.initials: M
authors.lastname: Maceroni
authors.forename: Margherita
authors.initials: M
authors.lastname: Eufemi
authors.forename: Fabio
authors.initials: F
authors.lastname: Altieri
mesh_terms: D019943:Amino Acid Substitution; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D015530:Nuclear Matrix / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human