Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase

In the mammalian endoplasmic reticulum, oxidoreductin-1α (Ero1α) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O; 2; to H; 2; O; 2; on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H; 2; O; 2; generation cause cell death, Ero1α activity must be properly regulated. In addition to the four catalytic cysteines (Cys; 94; , Cys; 99; , Cys; 104; , and Cys; 131; ) that are located in the flexible active site region, the Cys; 208; -Cys; 241; pair located at the base of another flexible loop is necessary for Ero1α regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys; 208; -Cys; 241; disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys; 208; and Cys; 241; did not grossly affect the thermal stability or overall shape of Ero1α, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys; 208; and Cys; 241; provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1α through reduction of the Cys; 208; -Cys; 241; disulfide. We propose a mechanism of dual Ero1α regulation by dynamic redox interactions between PDI and the two Ero1α flexible loops that harbor the regulatory cysteines.