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.
Highlights
ERp57, known as ERp60, ERp61, or GRP58, is a member of the protein-disulfide isomerases family, with PDI2 as the best-known example (1, 2)
It is mainly localized in the endoplasmic reticulum (ER) and shares with other family members a thiol-disulfide exchange activity strictly related to the redox properties of its thioredoxin-like active sites (2, 7–9)
To provide additional evidence of these observations and to determine the role in DNA binding control of the two cysteine residues present in the -CGHC- thioredoxin-like active site, we expressed in E. coli the aЈ domain and its C406S mutant without glutathione S-transferase tagging
Summary
Expression, and Purification of the C-terminal Domain of ERp57—The coding sequence of the ERp57 C-terminal region (residues 377–505) containing the aЈ domain was amplified from a plasmid containing the full-length cDNA of human ERp57, as previously described (33). Protein corresponding to the C406S mutant contained, as the wild type domain, a N-terminal pre-sequence coded by the pET29 vector and was expressed and purified as described above. Alkylation of Protein Samples with Iodoacetamide—Wild type C-terminal domain and C406S mutant (200 g) were denatured in 6 M guanidinium chloride, 0.25 M Tris-HCl, 1.25 mM EDTA, pH 7.0, in the absence of reducing agents, and quickly alkylated by the addition of iodoacetamide (1.1 M final concentration) at room temperature for 1 min, in the dark (37). Expression, and Purification of Human ERp57—The DNA fragment coding for mature human ERp57, devoid of the N-terminal pre-sequence (residues 1–24) and obtained by BamHI and EcoRI digestion of the pGEX-2T-ERp57 plasmid (33), was inserted into the BamHI and EcoRI sites of pTriEx-1.1 vector (Novagen) giving the pTriEx-1.1-ERp57 vector. Aliquots were analyzed by SDS-PAGE under non-reducing conditions and EMSA
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