A cysteine-based redox switch regulates millisecond dynamics of PDI and controls enzyme activity

Abstract

Protein disulfide isomerase (PDI) is a ubiquitous redox-regulated enzyme that interacts with hundreds of client proteins intracellularly and extracellularly. It comprises two redox-sensitive domains, each hosting the conserved catalytic motif CxxC, two redox-insensitive protein-binding domains, and three linkers. Snapshots of oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDIs structure dynamically changes in response to the redox microenvironment and ligand binding remain unknown. Here, we used multiparameter confocal single-molecule Forster resonance energy transfer (smFRET) and multiple FRET pairs to track the movements of the two catalytic domains with high temporal resolution. Our studies document 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 sub-millisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the two active sites are structurally nonequivalent and that ligands targeting the active sites of reduced PDI shift the equilibrium towards closed conformations of the enzyme. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize the multifaced role of PDI in biology and may assist drug development.