Studies On The Molecular Interaction Between Protein Disulfide Isomerase And Its Inhibitors

Protein disulfide isomerase(PDI)is a critical oxidoreductase in numerous physiological and pathological processes.Recently,PDI was found to play a vital role in thrombus formation in vivo and is recognized as a therapeutical target to prevent thrombosis.Rutin is one of potent inhibitors of PDI.We previously showed that rutin binds and inhibits PDI activities,leading to the suppression of platelet aggregation and fibrin generation in a mouse thrombosis model.However,the molecular mechanism of the interaction between rutin and PDI is still unclear despite extensive research efforts,including protein crystallization.Here,we investigated the molecular mechanism of the binding of rutin to PDI using a combination of experimental and computational techniques.We found that rutin can directly bind either human or murine PDI based on fluorescence binding assays.Our molecular docking and molecular dynamics simulations,complemented by site-directed mutagenesis and insulin reduction assays,provided a consistent picture that rutin binds at the interface between b'and x domains of PDI.The measurements of the interaction between PDI mutants and rutin by isothermal titration calorimetry(ITC)revealed that the residue H354,L355 and E359 affects this binding.Sequence analysis further indicates that the residues irnvolved in the binding of the inhibitor are not conserved across the extracellular thiol isomerases.which demonstrates that the ligand-binding site identified here is a potential target for the design of selective inhibitors for PDI.Furthermore,we identified three rutin analogues based on the active site found in this study,and evaluated the inhibitory activities of these analogues by insulin reduction assays,further confirming the validity of the current molecular model.Together,this work study the structural basis for the inhibitory mechanism of rutin,identify the active site of rutin binding to PDI,and provide a promising strategy for the design of novel inhibitors of PDI for therapeutic applications.