Molecular determinants of allosteric modulation of protein disulfide isomerase by small-molecule b′-ligands

Allosteric modulation is central to enzyme function and an attractive strategy for drug development. Protein Disulfide Isomerase (PDI), the prototypical thiol-isomerase, exemplifies this potential through its structural flexibility and involvement in neurodegeneration, cancer, and thromboinflammatory disorders such as sepsis, stroke, cancer-associated thrombosis, and antiphospholipid syndrome. PDI consists of four thioredoxin-like domains (a-b-b'-a'), with catalytic CGHC motifs in a and a' domains and a ligand-binding pocket in the b' domain. We previously reported that the b'-ligand bepristat 2a (Bep2a) inhibits PDI activity toward large macromolecular substrates while allosterically enhancing activity toward smaller physiological substrates such as GSSG and l-cystine. Here, we define the molecular, thermodynamic, and structural basis of this dual function. Bep2a features an indole ring with five substituents (R1-R5). Using mutagenesis and HDX-MS, we mapped the complex topology, identified five residues (F249, H256, I301, F304, I318) involved in binding, and uncovered a ligand-induced rearrangement of the left helix that acts as a dynamic gate controlling pocket accessibility, a previously unrecognized regulatory mechanism. AI-informed modeling, SAR analysis, and smFRET revealed that Bep2a's indole core binds perpendicularly in the pocket, with the R1 hydroxyl forming a critical hydrogen bond with H256, which is essential for binding but not for allosteric activation. Conversely, the R4 amine projects outward, serving as a key allosteric site that engages the catalytic domains and promotes PDI compaction. These findings uncover fundamental principles of PDI allosteric regulation and provide a blueprint for optimizing existing ligands and designing new ones with defined functional outcomes.