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.
Highlights
Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum
PDI mediates cleavage of disulfide bonds in glycoprotein 120 that are required for HIV-1 entry[18,19], and its inhibition interferes with the ability of HIV-1 to infect T cells[20]
Gene duplication resulted in the formation of multidomain enzymes in eukaryotes in which some thioredoxin-like domains retained catalytic function while others evolved into substrate-binding domains[33]
Summary
Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a0 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 activated by peptides and proteins and is present in other thiol isomerases. Peptides known to bind PDI, such as mastoparan and somatostatin and protein substrates (for example, cathespin G) triggered this substrate-driven allosteric switch These results support a general mechanism wherein ligation of the substrate-binding domain of PDI enhances its catalytic activity
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