Abstract
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.
Highlights
We found that Protein disulfide isomerase (PDI) can capture the hydrophobic segment of α -synuclein (α SN) primarily through its b′ domain and determined their binding mode in detail
The hydrophobic PDI-binding segment identified is involved in interactions with GroEL23 and PbaB24, suggesting that it displays a chaperone-philic binding motif that can be widely recognized as a mimic of the malfolded protein hallmarks
The α SN peptide contact site largely overlaps with the b′ surface involved in interactions with somatostatin and mastoparan, peptide inhibitors that compete with substrates, and with hydrophobic fluorescent probe ANS, which was previously characterized by nuclear magnetic resonance (NMR) chemical shift perturbation experiments[3,5,13]
Summary
In view of this situation, we attempted to provide the structural basis of PDI substrate recognition by using an appropriate model ligand. It has been reported that molecular chaperones actively contribute to the suppression of toxic aggregate formation of various amyloidogenic proteins associated with neurodegenerative disorders[19,20]. We have recently shown that α SN is capable of interacting with the bacterial chaperone GroEL23 and archaeal chaperone PbaB24, serving as a useful probe for characterizing their molecular recognition by biophysical techniques, which include nuclear magnetic resonance (NMR) spectroscopy and small-angle neutron scattering. We undertook to examine the possible interaction of PDI with α SN and, based on the results, we executed structural analyses using X-ray crystallography in conjunction with NMR spectroscopy that focused on the substrate-binding b′ –a′ domains of PDI
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