Vonrhein C, Flensburg C, Keller P, et al

Vonrhein C, Flensburg C, Keller P, et al. Data processing and analysis with the autoPROC toolbox. whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. Conclusions: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Rabbit Polyclonal to TAF1 Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of Meclofenoxate HCl small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions. as His-tagged small ubiquitin-like modifier (SUMO) fusion proteins29 using auto-induction ZYP-5052 media.30 Native proteins were prepared by treating the fusion proteins with SUMO hydrolase and purified by subtractive immobilized metal affinity chromatography, ion exchange chromatography, and gel filtration at 4C. 2.2 O. Crystallization and data collection To prepare the PAI-1-stab/Nb42 complex, PAI-1-stab was incubated with Nb42 (1:1.1 molar ratio, 22C, 30 minutes) and concentrated to 5.6 mg mL?1 at 4C. The complex was crystallized using the sitting-drop vapor diffusion method, mixing an equal volume of protein solution with precipitant (1 mol L?1 succinic acid, 0.1 mol L?1 HEPES, 1% w/v PEG2000, pH 7.0) at 20C. For cryoprotection, the drop was overlaid with paraffin oil and the crystals were pulled through the oil before flash-cooling in liquid nitrogen. The triple PAI-1-stab/Nb42/Nb64 complex (1:1.1:1.1 molar ratio) was prepared in a similar way as the PAI-1-stab/Nb42 complex and concentrated to 6.8 mg mL?1. Optimized crystals were grown by mixing the protein solution with precipitant (0.1 mol L?1 Bis-Tris pH 6.5, 17% w/v PEG3350, 3% v/v methanol) at a 2:1 volume ratio in a sitting-drop vapor diffusion format at 20C and cryoprotected by transferring the crystals through paraffin oil. The PAI-1-W175F/Nb42/Nb64 complex was prepared in a similar way as PAI-1-stab/Nb42/Nb64 and concentrated to 6.8 mg mL?1. Optimized crystals were grown by mixing the protein solution with precipitant (0.1 mol L?1 Bis-Tris pH 6.5, 10% w/v PEG3350) at a volume ratio of 2:1 using hanging-drop vapor diffusion at 4C. Crystals were cryoprotected by briefly transferring the crystal to crystallization solution supplemented with 20% v/v ethylene glycol. X-ray diffraction data were collected at 100 Meclofenoxate HCl K using the PROXIMA2 beamline of SOLEIL Synchrotron and the ID-30A and ID-30B beamlines at the European Synchrotron Radiation Facility (ESRF). Data collection and refinement statistics are listed in Table 1. TABLE 1 Meclofenoxate HCl Data collection and refinement statistics 2121(?)45.48, 71.42, 96.6043.68, 70.80, 98.5189.87, 123.65, 64.98??,, ()90, 100.54, 9090, 97.48, 9090, 129.94, 90?Resolution (?)94.97-2.03 (2.05-2.03)48.84-2.28 (2.34-2.28)49.82-2.32 (2.35-2.32)?factors (?2)??Protein55.8961.4355.09??Water51.8749.2451.91?R.m.s. deviations??Bond lengths (?)0.0070.0090.009??Bond angles () Open in a separate window Diffraction data were collected from a single crystal. The values in parentheses are for the highest resolution shell. 2.3 O. Structure determination, refinement, and analysis The obtained diffraction data were processed using autoPROC in default settings, with a high-resolution cutoff on 0.60.31 The data were initially phased by molecular replacement using the structure of PAI-1-W175F (Protein Data Bank [PDB]: 3Q0232) or PAI-1-stab (PDB: 1DB233) in active conformation as a search model using PHASER.34 Nb42 and Nb64 were initially modelled based on homologous Nb structures that were selected using a protein fundamental local alignment search tool (BLAST) (PDB: 5JA8 and 5JA9, respectively35). The constructions of the PAI-1/Nb complexes were improved by iterative rounds of manual rebuilding in Coot and refinement in phenix.refine.36 Final models have been deposited to the PDB under the accession codes 6GWN (PAI-1-W175F/Nb42/Nb64), 6GWP (PAI-1-stab/Nb42/Nb64), and 6GWQ (PAI-1-stab/Nb42). PyMOL (The PyMOL Molecular Graphics System, version 2.0.7, Schr?dinger, LLC) was used to visualize and superimpose models and to compute root-mean-squared deviations for those C atoms (C RMSD). The interfaces present in the complexes were analyzed using the PISA software.37 2.4 O. Size exclusion chromatography and inline small-angle X-ray scattering (SAXS) SAXS experiments were performed at 288 K within the PAI-1/Nb complexes inside a buffer comprising 20 mmol L?1 Bis-Tris pH Meclofenoxate HCl 6, 300 mmol L?1 NaCl and 5% v/v glycerol in the SWING beamline (SOLEIL Synchrotron). Protein samples (40-50 L, ~10 mg mL?1) were loaded on an Agilent Bio SEC-3, 300?, 4.6 300-mm high-performance liquid chromatography column and were eluted into the SAXS capillary cell at a flow rate of 0.3 mL min?1. SAXS data for both buffer (180 frames, 750 ms exposure time, and 250 ms deceased time) and PAI-1/Nb complex (254 frames, same exposure and dead instances) were collected. Buffer frames were processed, averaged, and subtracted from each protein framework (excluding outliers) using Scatter.38 For each sample, five scattering curves collected round the maximum maximum were selected using CORMAP39 and averaged and scaled using Meclofenoxate HCl Scatter.38 Solution-scattering profiles from your atomic structures of the complexes were calculated and fitted to the experimental averaged scattering curves using CRYSOL.40 Models for fitting the SAXS.

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