Abstract
Protein-disulfide isomerase (PDI), an endoplasmic reticulum (ER)-resident protein, is primarily known as a catalyst of oxidative protein folding but also has a protein unfolding activity. We showed previously that PDI unfolds the cholera toxin A1 (CTA1) polypeptide to facilitate the ER-to-cytosol retrotranslocation of the toxin during intoxication. We now provide insight into the mechanism of this unfoldase activity. PDI includes two redox-active (a and a') and two redox-inactive (b and b') thioredoxin-like domains, a linker (x), and a C-terminal domain (c) arranged as abb'xa'c. Using recombinant PDI fragments, we show that binding of CTA1 by the continuous PDIbb'xa' fragment is necessary and sufficient to trigger unfolding. The specific linear arrangement of bb'xa' and the type a domain (a' versus a) C-terminal to bb'x are additional determinants of activity. These data suggest a general mechanism for the unfoldase activity of PDI: the concurrent and specific binding of bb'xa' to particular regions along the CTA1 molecule triggers its unfolding. Furthermore, we show the bb' domains of PDI are indispensable to the unfolding reaction, whereas the function of its a' domain can be substituted partially by the a' domain from ERp57 (abb'xa'c) or ERp72 (ca degrees abb'xa'), PDI-like proteins that do not unfold CTA1 normally. However, the bb' domains of PDI were insufficient to convert full-length ERp57 into an unfoldase because the a domain of ERp57 inhibited toxin binding. Thus, we propose that generating an unfoldase from thioredoxin-like domains requires the bb'(x) domains of PDI followed by an a' domain but not preceded by an inhibitory a domain.
Highlights
Erone, binding polypeptides to prevent their aggregation [11,12,13]
Recombinant Protein-disulfide isomerase (PDI) Unfolds cholera toxin A1 (CTA1) Efficiently—Native PDI derived from bovine liver was shown previously to unfold the catalytic A subunit (CTA) and CTA1 polypeptides, rendering them sensitive to tryptic digestion [20]
Whereas CTA1 was mostly resistant to proteolysis when incubated with BSA (Fig. 1B, compare lane 2 with 1), it became sensitive to tryptic digestion when incubated with PDI
Summary
Erone, binding polypeptides to prevent their aggregation [11,12,13]. PDI acts as a structural subunit of the prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein complexes; this function is similar to its chaperone activity (14 –19). In addition to suggesting a general mechanism for the unfoldase activity of PDI, our data indicate functional similarities and differences among thioredoxin-like domains of PDI family proteins.
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