Protein design, expression, and purification
Truncated, His-tagged, fully functional versions of human CYP2A6, CYP2A6 I208S/G301A/I300F/S369G, CYP2A13 and CYP2E1 were designed, expressed and purified as described previously [5,12].
Spectral binding assay
Ligand-binding affinities were determined with a previously described spectral-ligand binding assay .
All metabolism and inhibition assays used a reconstituted protein system (RPS) consisting of 50 pmol of purified CYP2A or CYP2E1 incubated with 200 pmol of NADPH-cytochrome P450 reductase and 100 pmol of cytochrome b5 in a 1:4:2 ratio for 20 min at room temperature prior to use. This RPS mixture was added to assay buffer containing the desired substrate and inhibitor. The samples were preincubated at 37 °C for 3 min, and the reactions were initiated by the addition of 1 mm NADPH. Samples were incubated for 10 min at 37 °C, and the reaction was stopped with 300 μL of 20% trichloroacetic acid (TCA) on ice. All standards and zero samples had 300 μL of 20% TCA added prior to the addition of NADPH. Samples and standards were centrifuged at 4500 g for 10 min.
RPS was added to buffer (50 mm Tris, pH 7.4, and 5 mm MgCl2) containing coumarin and pilocarpine. After completion of the assay as described above, all samples and standards were diluted by the addition of 1 mL (CYP2A6) or 200 μL (CYP2A13) of buffer (50 mm Tris, pH 7.4, and 5 mm MgCl2). The amount of 7-hydroxycoumarin present was determined by fluorescence following HPLC separation. The 7-hydroxycoumarin metabolite was detected by fluorescence with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The mobile phase consisted of 30% MeOH, 68% water, and 2% acetic acid, with a 1 mL·min−1 flow rate and a sample volume of 10 μL.
RPS was added to 100 mm potassium phosphate (pH 6.8) with 2 mm ascorbic acid (for CYP2E1), p-nitrophenol, and pilocarpine. After assay completion as described above, the amount of 4-nitrocatechol was determined following HPLC separation. A mobile phase of 27% acetonitrile and 0.2% acetic acid was run at a rate of 1 mL·min−1. Monitoring of absorption at 345 nm allowed the detection of 4-nitrocatechol at ∼ 5 min, and p-nitrophenol at ∼ 8 min.
RPS was added to 100 mm potassium phosphate buffer (pH 7.4) and varying concentrations of chlorzoxazone and pilocarpine. Sample reactions were stopped after 10 min at 37 °C by the addition of 25 μL of 60% perchloric acid, instead of TCA. Corresponding standards and controls had 25 μL of 60% perchloric acid, instead of TCA, added prior to the addition of NADPH. The 6-hydroxychlorzoxazone metabolite was detected with an absorption wavelength of 287 nm by HPLC with a mobile phase consisting of 20% acetonitrile, 78% water, and 2% acetic acid, with a 1 mL·min−1 flow rate.
Protein crystallization, data collection, and structure determination
CYP2A6, CYP2A6 I208S/I300F/G301A/S369G, CYP2A13 and CYP2E1 were cocrystallized with pilocarpine by hanging drop vapor diffusion. The CYP2A6–pilocarpine crystals were grown from 500 μm CYP2A6 with 100 mm pilocarpine in CM elution buffer (50 mm potassium phosphate buffer, pH 7.4, 20% glycerol, 1 mm EDTA, 0.5 m NaCl) with 2% Anapoe-35 in a 1:1 ratio with precipitant solution [30% poly(ethylene glycol) 3500, 0.175 m Tris, pH 8.5, 0.2 m ammonium sulfate]. The CYP2A6 I208S/I300F/G301A/S369G–pilocarpine crystal was grown similarly, with 100 μm CYP2A6 I208S/I300F/G301A/S369G and 100 mm pilocarpine in CM elution buffer and 2% Anapoe-35 in a 1:1 ratio with a slightly modified precipitant solution [30% poly(ethylene glycol) 3350, 0.100 m Tris, pH 8.5, 0.200 m ammonium sulfate]. The CYP2A13–pilocarpine crystal was grown from a solution of 200 μm CYP2A13, 100 mm pilocarpine and 2% Anapoe-35 in CM elution buffer in a 2:1 ratio with precipitant solution [30% poly(ethylene glycol) 3350, 0.175 m Tris, pH 8.5, 0.2 m ammonium sulfate]. The CYP2E1 crystal was grown at 455 μm in a buffer containing 100 mm pilocarpine, 120 mm potassium phosphate (pH 7.4), 0.5 m sucrose and 1 mm EDTA in a 1:1 ratio with precipitant solution [8% poly(ethylene glycol) MME 2000, 12% isopropanol, 0.1 m NaHEPES, pH 7.5].
Crystals were flash cooled in liquid nitrogen after being immersed in a cryoprotectant. For all CYP2A crystals, the cryoprotectant consisted of 700 μL of synthetic mother liquor and 300 μL of 100% ethylene glycol. The cryoprotectant for CYP2E1 was 0.1 m NaHEPES (pH 7.5), 5% isopropanol, and 1.4 m sucrose. X-ray diffraction data for CYP2A6, CYP2A6 I208S/I300F/G301A/S369G, and CYP2A13, each with pilocarpine, were collected at the Stanford Synchrotron Radiation Laboratory (Stanford, CA, USA) on Beamline 9-2 with a 0.98-Å wavelength and temperature of 100 K. Data were processed with mosflm and scala [16,17]. The CYP2E1–pilocarpine dataset was collected on Beamline 17-BM at the Advanced Photon Source and processed with hkl2000. All structures were solved by molecular replacement with phaser . Model building and refinement of all structures were performed iteratively with coot  and refmac5 in the ccp4 suite . Detailed collection and refinement statistics are given in Table 2.
Table 2. Crystal data collection and refinement statistics of CYP enzymes binding pilocarpine.
| Space group||P21||P21||P1||P4|
| Unit cell|
| a, b, c (Å)||70.34, 158.00, 104.45||70.95, 159.8, 103.9||71.50, 119.86, 154.87||100.6, 100.6, 259.5|
| α, β, γ (°)||90.0, 92.1, 90.0||90.0, 91.8, 90.0||101.0, 101.7, 93.6||90.0, 90.0, 90.0|
| Molecules per AU||4||4||8||4|
| Resolution (Å)||87.04–2.40 (2.46–2.40)||87.04–2.10 (2.16–2.10)||117.00–3.00 (3.16–3.00)||37.0–2.35 (2.41–2.35)|
| Total observationsa||237 442 (17 456)||301 406 (21 586)||383 997 (55 808)||385 690 (18 326)|
| Unique observationsa||85 624 (6420)||126 622 (9537)||96 716 (14 053)||102 444 (10 635)|
| Completenessa (%)||96.5 (97.9)||94.3 (96.0)||98.3 (97.9)||95.9 (84.3)|
| Multiplicitya||2.8 (2.7)||2.4 (2.3)||4.0 (4.0)||3.76 (2.04)|
| Rmergea (%)||0.075 (0.400)||0.075 (0.400)||0.117 (0.634)||0.105 (0.221)|
| I/σI a||13.2 (3.4)||9.3 (1.4)||9.4 (2.1)||6.6 (2.6)|
| Resolution (Å)||79.00–2.40||63.32–2.10||102.88–3.00||36.98–2.35|
| No. of reflections||81 313||120 218||91 882||97 337|
| R/Rfree (%)||19.9/26.0||21.1/25.7||21.3/30.3||21.7/28.1|
| Rmsd bond lengths (Å)||0.020||0.009||0.017||0.020|
| Rmsd bond angles (°)||1.847||1.182||1.786||1.629|
|Number of atoms/average B-factor|
| Protein||15 040/31.8||15 101/31.5||30 058/55.4||15 166/35.4|
| Coordinate error, Luzzati plot (Å)||0.277||0.254||0.388||0.321|
The search model for CYP2A13 with pilocarpine was a 1.65-Å structure of CYP2A6 with N-methyl[5-(pyridin-3-yl)furan-2-yl]methanamine [Protein Data Bank (PDB) 2FDV]. A Matthews coefficient of 2.88 with 57.4% solvent suggested eight molecules in the asymmetric unit. Molecular replacement identified eight molecules in CYP2A13 with good packing. The 3.0-Å final model of CYP2A13 with pilocarpine contains residues 32–494 in molecules A–H, heme, six pilocarpine molecules, and 31 water molecules, and is deposited in the PDB as 3T3S. The crystallographic R-factor is 21.3% and the Rfree is 30.3%. In the Ramachandran plot, 86.2% of the residues are in the most favored region, 13.0% in the additionally allowed region, 0.5% in the generously allowed region, and 0.3% in the disallowed region.
The search model for both CYP2A6 structures was a 1.9-Å structure of CYP2A6 complexed with coumarin (PDB 1Z10, molecule B). The 2.4-Å structure of CYP2A6 with pilocarpine contains residues 32–494 and heme in all four copies of the asymmetric unit, four pilocarpine molecules, and 217 water molecules, and is deposited in the PDB as 3T3R. The crystallographic R-factor is 19.9% and the Rfree is 26.0%. In the Ramachandran plot, 91.5% of the residues are in the most favored region, 8.2% in the additionally allowed region, 0.4% in the generously allowed region, and 0.2% in the disallowed region.
The 2.1-Å final model for CYP2A6 I208S/I300F/G301A/S369G with pilocarpine contains residues 32–494 and heme in all four molecules, four pilocarpine molecules, and 504 water molecules, and is deposited in the PDB as 3T3Q. The crystallographic R-factor is 21.2% and the Rfree is 25.7%. In the Ramachandran plot, 91.2% of the residues are in the most favored region, 8.1% in the additionally allowed region, 0.3% in the generously allowed region, and 0.2% in the disallowed region.
The 2.35-Å final model of CYP2E1 with pilocarpine contains residues 31–494, heme and pilocarpine in all four molecules. There are also four sucrose molecules and 772 water molecules, and it is deposited in the PDB as 3T3Z. The crystallographic R-factor is 21.7% and the Rfree is 28.1%. In the Ramachandran plot, 89.6% of the residues are in the most favored region, 9.9% in the additionally allowed region, 0.2% in the generously allowed region, and 0.2% in the disallowed region.
In each structure, the single residue found in the disallowed region of the Ramachandran plot was well defined by electron density.
Protein figures and analysis
All protein figures were generated with pymol . voidoo  was used to determine active site volumes with a 1.4-Å probe size. All figures were made with molecule A for CYP2A6, CYP2A6 I208S/I300F/G301A/S369G, and CYP2E1. All figures of CYP2A13 utilized molecule E, as this molecule contained the best ligand density.