Mechanistic basis of activation and inhibition of Protein Disulfide Isomerase by allosteric antithrombotic compounds.

Background: Protein Disulfide Isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development.

Objectives: To define the mechanism of action of two allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a.

Methods: A multi-pronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single turnover kinetics, and site-specific mutagenesis.

Results: PDI is a thiol-isomerase consisting of two catalytic a-domains and two inactive b-domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of nine FRET sensors, we show that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, FRET data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme-substrate complex.

Conclusions: This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a, and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.