Computational protocol: Structural basis of redox-dependent substrate binding of protein disulfide isomerase

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[…] NMR measurements were performed in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM KCl, and 10% (v/v) D2O using an AVANCE800 spectrometer (Bruker Biospin) equipped with a 5 mm triple-resonance cryogenic probe. To prepare the reduced form of the PDI proteins, 10 mM d-DTT was added to the buffer. The 1H-15N HSQC spectra were recorded at a 1H observation frequency of 800.32 MHz with 256 (t1) × 2048 (t2) complex points. The spectral data of the PDI-derived proteins (at a concentration of 0.05 mM) were acquired at 303 K in the presence and absence of 0.2 mM full-length αSN or 0.2 mM αSN peptide. The spectral assignments of the PDI b′–a′ domains, b′ domain, and a′ domain have been described previously. The HSQC spectra of 15N-labeled full-length αSN (at a concentration of 0.05 mM) were measured at 283 K in the presence and absence of 0.01–0.05 mM PDI b′–a′ domains. The NMR assignments of αSN have been described previously. Chemical shift perturbations were quantified as (0.04ΔδN2 + ΔδH2)1/2, where ΔδH and ΔδN are the observed chemical shift changes for 1H and 15N, respectively. The NMR data were processed and analyzed using TOPSPIN-2.1 (Bruker Biospin) and SPARKY software. In NMR perturbation profiles, proline residues and the residues whose 1H-15N HSQC peaks could not be observed because of peak overlapping and/or broadening were shown by asterisks. [...] The crystals of the PDI b′–a′ domains (10 mg/ml) complexed with αSN peptide (1:5 molar ratio) were grown in 0.1 M HEPES buffer (pH 7.5) containing 25% (w/v) PEG3350 for a week at 293 K. The crystals were directly transferred into the reservoir solution and flash-cooled in liquid nitrogen. The diffraction data set was collected using synchrotron radiation at BL44XU of SPring-8 (Japan), and was scaled and integrated using HKL2000. Crystal parameters are summarized in .The 1.60-Å resolution crystal structure of the PDI b′–a′ domains complexed with the αSN peptide was solved by molecular replacement using the program MOLREP with the isolated b′ and a′ domain coordinates derived from the crystal structure of H. insolens PDI b′–a′ domain (oxidized form, 3WT2) as search models. Model building into the electron density maps and refinement were performed using COOT and REFMAC5, respectively. The stereochemical quality of the final model was validated by PROCHECK. The final refinement statistics are summarized in . Molecular graphic figures were prepared using PyMOL (http://www.pymol.org/). […]

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