The Human Cyclophilin Family of Peptidyl-Prolyl Isomerases - Davis et al.

Material and Methods

Cloning, Expression, and Purification of Isomerase Domains: Detailed materials and methods for cloning, expression, purification, and crystallization of all novel isomerase domain structures solved as part of the Structural Genomics Consortium are freely available at the website http://www.sgc.utoronto.ca/ , and where methods differ significantly from the following they are noted for each isoform in the supplemental methods. In general, full-length cDNA clones were obtained from the Mammalian Gene Collection (accession numbers noted above). Constructs based around the predicted isomerase domain boundaries were cloned into pET28a-LIC using ligation independent cloning methods (BD Biosciences, San Jose, CA, USA) and transformed into BL21 Gold (DE3) cells (Stratagene, La Jolla, CA, USA). The resulting vectors encode an N-terminal (His)6 tag with a thrombin cleavage site. Cultures were grown in Terrific Broth medium at 37 °C to OD600=6, and induced at 15 °C overnight with the addition of 50-100 µm isopropyl thio-b-d-galactoside (IPTG). Pellets were resuspended in 20 mL of lysis buffer (50 mm Tris, pH 8.0, 500 mm NaCl, 1 mm phenylmethanesulfonyl fluoride (PMSF) and 0.1 mL general protease inhibitor (Sigma P2714, St Louis, MO, USA) and lysed by sonication; lysates were then centrifuged for 20 min at 69, 673 g. The supernatant was loaded onto nickel nitrilotriacetic acid resin (Ni-NTA) (Qiagen, Valencia, CA, USA), washed with five column volumes of lysis buffer and five column volumes of low imidazole buffer (lysis buffer + 10 mm imidazole, pH 8), and eluted in 10 mL of elution buffer (lysis buffer + 250 mm imidazole, pH 8, and 10% glycerol). If the (His)6 tag was cleaved for crystallization purposes, then one unit of thrombin (Sigma) per milligram of protein was added to remove the tag overnight at 4 °C. For gel filtration, an XK 16 x 65 column (GE Healthcare, Piscataway, NJ, USA) packed with HiLoad Superdex 200 resin was pre-equilibrated with gel filtration buffer (lysis buffer + 5 mm b-mercaptoethanol and 1 mm EDTA). Peak fractions were pooled and concentrated using Amicon concentrators (10,000 molecular mass cut-off; Millipore, Danvers, MA, USA). The protein was generally used at 250-500 µM for crystallization screening.

Crystallization and Structure Solution of Isomerase Domains: Generally, crystal hits were initially prepared in sitting drop 96-well format. Proteins were set up as 1 µL protein + 1 µL reservoir solution and incubated at 18 oC for 24 hours-1 month. If crystal optimization was required it was performed in 24-well hanging drop format with 1 µL protein + 1 µL reservoir solution. Crystals were cryoprotected with mother liquor with 10-15% glycerol. Datasets were collected on an in-house FR-E SuperBright Cu rotating anode / Raxis IV++ detector (Rigaku Americas, The Woodlands, TX, USA), except for PPIC which was collected at APS 19-BM. Data was usually integrated and scaled using the HKL2000 program package [59,60]. For structure solution and refinement, the program PHASER [61] was used as part of the CCP4 suite [62] to find the molecular replacement solution. Manual rebuilding was performed using either O [63] or COOT [64], and refined using REFMAC [65] in the CCP4I program suite [66]. In most cases ARP/wARP was utilized to assist in model building and iterative refinement of starting phases [67]. Final models were evaluated using PROCHECK [68] and MOLPROBITY [69], with all models judged to have excellent stereochemistry and no residues in disallowed regions of Ramachandran space. PPIC. Optimized PPIC crystals were obtained using the hanging drop vapor diffusion method. Crystals grew when the protein (encoding residues at 15 mg/mL was pre-incubated with cyclosporin A in a 1:2 molar ratio for at least overnight and then mixed with the reservoir solution in a 1:1 volume ratio. The drop was equilibrated against a reservoir solution containing 25% PEG MME 550, 0.1 M zinc acetate, 0.1 M MES at pH 6.5. PPIE. Diffracting crystals leading to the structure grew when the protein was mixed at 20 mg/mL with the reservoir solution (containing 34% PEG 8K, 0.2M NH4SO4, and 0.1M Bis-Tris pH 6) in a 1:1 volume ratio. PPIG. Purified PPIG K125A/E126A was crystallized using the sitting drop vapor diffusion method at 18 °C by mixing 0.2 µl of the protein solution with 0.2 µl of the reservoir solution containing 2M ammonium sulfate, 0.2M Sodium chloride, 0.1M Hepes, pH 7.5. PPIL2. Diffracting crystals leading to the structure grew when the protein was mixed at 20 mg/mL with the reservoir solution (containing 0.8M Potassium Sodium Tartrate tetrahydrate, 0.1M Hepes pH 7.5) in a 1:1 volume ratio. PPWD1. Purified PPWD1 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (12 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 1.7 M ammonium sulfate, 0.1 M sodium cacodylate, 0. 2M sodium acetate at pH 5.7. Full methods can be found in [29]. NKTR. Crystals grew in hanging drop format when protein at 15 mg/mL was mixed with reservoir containing 21% Peg 3350, 0.25M potassium sulfate in a 1:1 ratio. SDCCAG-10. Crystals were obtained when the protein at 20 mg/mL was mixed with reservoir solution containing 20% Peg 3350 and 0.2M NaI in 1:1 ratio in hanging drop format.

Thermal Stabilization Assay: All protein samples used for static light scattering (StarGazer) trials were assessed for purity utilizing SDS-PAGE and verified for mass accuracy using mass spectrometry. Methods were generally as described as in [36]; protein at approximately 20 µM concentration was heated from room temperature to 80 oC either in the presence or absence of small molecules, including cyclosporins A, C, D, and H (LKT Labs, MN, USA). Ligand binding was detected by monitoring the increase in Tagg in the presence of the ligand; and any compound that caused a >2°C increase in Tagg were observed to be outside of the range of experimental error. Each compound was tested at least twice.

NMR-based Activity Assay: All protein samples aimed at assessing binding and/or catalysis of tetrapeptide substrates were diluted to 500 µL with 10% D2O and placed into a Shigemi microcell (Allison Park, PA, USA). Samples contained 1 mm protein and either 0.075 mm suc-AAPF-pNA, suc-AFPF-pNA, or suc-AGPF-pNA (Bachem), along with 100 mM phosphate buffer pH 7 and 100 mM NaCl. Spectra were collected at 10 °C on a Varian 600 or 900 MHz spectrometer (Palo Alto, CA, USA). Spectra were acquired using standard Varian BioPack sequences, processed using NMRpipe software [70] and visualized using ccpn software [71].

Monte Carlo Simulations. A set of 400 test peptides of the general form X-Z-Gly-Pro were docked to a subset of cyclophilin isoforms (PPIA - PDB: 1AK4, PPIL2 - PDB: 1ZKC, PPIC- PDB : 2ESL, PPWD1 - PDB: 2A2N, and NKTR - PDB: 2HE9) using the ICM software (Molsoft LLC). Monte Carlo simulations were performed to sample conformational space for each combination of cyclophilin isoform and test peptide, allowing flexibility of the tetrapeptide and the sidechains of the gatekeepers at positions comparable to PPIA Thr73, Lys82, and Ala103, and keeping the rest of the receptor rigid [55]. The crystal structure of PPWD1 (PDB: 2A2N) was used to determine the initial position of each tetrapeptide in the various cyclophilin isoforms by superimposing the Gly and Pro residues onto the corresponding residues bound to the active site of PPWD1; and the catalytic arginine was repositioned to align with Arg535 of PPWD1. Throughout the Monte Carlo simulations (200,000 iterations), tethers were imposed on the C-terminal Gly and Pro residues, to ensure that the tetrapeptides would remain bound to the active site. For each combination of cyclophilin isoform and tetrapeptide, the lowest energy complex was chosen as the predicted conformation of the bound complex, and an estimate of the binding energy was calculated using ICM (Molsoft, LLC) [56]. Additionally, the distance between the tetrapeptide and the Cα of the gatekeeper equivalent to PPIA Lys82 was calculated (this residue is located at the far end of the -2 pocket, opposite to the active site pocket, as shown in Figure 5), to determine how well the docked peptide was predicted to fill the -2 pocket.

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