Substrate-Induced Unfolding of Protein Disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from Its Holotoxin

To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation. Author Summary Protein disulfide isomerase (PDI) is a luminal endoplasmic reticulum (ER) protein with related but independent oxidoreductase and chaperone activities. The molecular mechanism of PDI chaperone function remains unidentified. Here, we report that PDI unfolds upon contact with the catalytic A1 subunit of cholera toxin (CT). This unfolding event dislodges CTA1 from the rest of the multimeric toxin, which is a prerequisite for the ER-to-cytosol export of CTA1 and toxin activity against the host cell. The substrate-induced unfolding of PDI is linked to its chaperone activity. Our work has established a new property of PDI that is required for CT disassembly and provides a possible structural basis for the broader role of PDI as a chaperone that prevents protein aggregation.