Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles

J Biol Chem. 2022 Aug;298(8):102217. doi: 10.1016/j.jbc.2022.102217. Epub 2022 Jun 30.


Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a') and two inactive (b, b'), organized to form a flexible abb'a' U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI's structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2, and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2 and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.

Keywords: allostery; protein disulfide isomerase; protein dynamics; single-molecule FRET; thiol isomerases; thrombosis.

MeSH terms

  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Humans
  • Oxidation-Reduction
  • Protein Disulfide-Isomerases* / metabolism
  • Protein Folding*


  • Protein Disulfide-Isomerases
  • Cysteine