A substrate-driven allosteric switch that enhances PDI catalytic activity

Abstract

Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a–b–b′–x–a′, wherein the thioredoxin-like a and a′ domains mediate disulfide bond shuffling and b and b′ domains are substrate binding. The b′ and a′ domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b′. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a′ by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains.

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

“…PDI, which may enable a more targeted investigation of the thrombotic mechanisms under regulatory control of PDI in vivo (32,33). PDI activity has previously been shown to influence platelet-dependent thrombin generation, which we now show is dependent on PDI-mediated activation of platelet factor V. Although platelet-derived factor Va only accounts for 20% of the total amount of factor V measured in plasma, this pool plays a critical role in supporting hemostasis.…”

Vascular thiol isomerases

“…Recent studies show that on binding a substrate, PDI assumes a more compact conformation. 25 Substrate binding at the hydrophobic pocket activates an allosteric switch involving the x-linker, which results in enhanced reductase activity in the a and a9 domains and appears to be a cooperative mechanism whereby substrate engagement activates enzymatic activity.…”

Advances in vascular thiol isomerase function

“…Bekendam et al demonstrated an allosteric switch mechanism, whereby binding of peptides or proteins to the hydrophobic binding site on the b' domain of PDI displaces the x -linker [17]. This displacement results in a conformational change in PDI, causing the structure to become more compact (Fig.…”

“…1). Differential cysteine alkylation coupled with mass spectroscopy showed an increase in active site cysteine thiolates and an increase in the reductase activity of PDI in both the a and a' domains [17]. Thus, activation of the allosteric switch appears to enhance reductase activity.…”

“…Two of these compounds were found to act at the hydrophobic binding pocket of the b' domain and were therefore termed bepristats [17]. Unlike flavonoid quercetins, however, whose binding to b' blocks reductase activity at the active site motifs, bepristats paradoxically enhanced reductase activity.…”