Molecular Bases of Cyclic and Specific Disulfide Interchange between Human ERO1α Protein and Protein-disulfide Isomerase (PDI)

Abstract

In the endoplasmic reticulum (ER) of human cells, ERO1␣ and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1␣ is essential to avoid ER hyperoxidation. To investigate how ERO1␣ oxidizes PDI selectively among more than 20 ERresident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding ␤-hairpin of ERO1␣ specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a-domain. ERO1␣ associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1␣ to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1␣ with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1␣-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1␣ activity may suggest its physiological role in ER redox and protein homeostasis.

Functional in Vitro Analysis of the ERO1 Protein and Protein-disulfide Isomerase Pathway

“…SPR analysis using point mutations in the bЈ domain of PDI suggested that it is the bЈ domain that contributes to the specific binding with ERO1␣, a result that is consistent with previous reports (Fig. 4B) (11,25,33). If the single domains of each a and aЈ domain were extracted, they showed the same oxygen consumption rate in the presence of constitutively active ERO1␣ (11).…”

“…Domain-swapping mutants of PDI showed that the position of aЈ domain was preferentially oxidized, suggesting the importance of the domain position (25). Recent structural analysis indicates that the bЈ domain of PDI specifically interacts with a protruding ␤-hairpin of ERO1␣ (33). Hence, the specific binding site and domain architecture of PDI allow specific binding between PDI and ERO1␣ and define the preferred oxidation of the aЈ domain by ERO1␣.…”

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

“…truding ␤-hairpin loop in Ero1␣ and the substrate binding pocket in the PDI b domain and via a sustained thiol-disulfide exchange between the loop I cysteines of Ero1␣ and the a domain active site of PDI during catalysis (32). More recently, however, we found that a non-catalytic mixed disulfide complex involving Ero1␣ Cys 208 or Cys 241 (Ero1␣-PDI fast ) is the predominant species detectable in the ER at steady state (18).…”

The Physiological Functions of Mammalian Endoplasmic Oxidoreductin 1: On Disulfides and More

“…It has been conclusively demonstrated that these interactions are of hydrophobic nature (29). They involve a protruding b-hairpin in Ero1a, which contains a critical tryptophan residue at its very tip (Trp 272 ), and a hydrophobic cleft in the substrate-binding domain of PDI (36). Even though experimental data were exclusively generated with Ero1a, both the hairpin structure and the crucial tryptophan are present in Ero1b as well (Fig.…”

“…Accordingly, the principle of substrate recognition is probably conserved among the mammalian isoforms. In addition, as hinted by in silico complex modeling (36), this mode of interaction presumably facilitates the specific thiol-disulfide exchange between the Cterminal active-site domain of PDI and the shuttle disulfide in Ero1a (7, 9, 10, 15, 52). In contrast, Ero1p harbors no tryptophan-containing b-hairpin (25) and preferentially oxidizes the N-terminal active-site domain of yeast PDI (51).…”