Structural basis of redox-dependent substrate binding of protein disulfide isomerase

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b ′ and a ′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a ′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b ′– a ′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b ′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a ′ domain partially masks the binding surface of the b ′ domain, causing steric hindrance against the peptide in the reduced form of the b ′– a ′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI.