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Originally published In Press as doi:10.1074/jbc.M405427200 on June 4, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35630-35637, August 20, 2004
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The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-Å Resolution*

Michael R. Wester{ddagger}§, Jason K. Yano{ddagger}§, Guillaume A. Schoch{ddagger}, Christine Yang{ddagger}, Keith J. Griffin{ddagger}, C. David Stout¶||, and Eric F. Johnson{ddagger}**

From the Departments of {ddagger}Molecular and Experimental Medicine and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, May 17, 2004 , and in revised form, June 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
P450 2C9 is one of three human microsomal cytochrome P450s (CYPs)1 in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The P450 2C9 locus is polymorphic leading to a diminished capacity to clear specific drugs in genetically affected individuals. For P450 2C9 substrates, such as warfarin or phenytoin, that have low therapeutic margins of safety, diminished metabolic capacity because of genetic polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses (1). P450 2C9 has also been implicated in the synthesis of arachidonic acid epoxides in extrahepatic tissues where they regulate blood pressure (2). Like other P450 subfamilies, the 2C enzymes share roughly 70% or greater amino acid identity. However, the 2C genes have duplicated and diverged rapidly as mammalian species evolved, leading to different numbers of enzymes in various species and highly divergent substrate selectivities. This diversity reflects high rates of non-synonymous substitutions that often alter residues that line the active site cavity and determine substrate selectivity.

Human P450s 2C9 and 2C19 are closely related with roughly 91% amino acid identity. Although they exhibit distinct substrate selectivities, residues predicted to line the active site cavity, based on the published structures of other mammalian P450s (3–6), do not differ between the two enzymes. This suggests that conformation changes are likely to underlie differences in the substrate selectivities of P450s 2C9 and 2C19 and that the structure(s) of one or both will differ from those published previously. This is supported by studies of chimeric enzymes generated from P450s 2C9 and 2C19 that have generally identified amino acid residues that are predicted to reside outside the substrate binding cavity as determinants of their distinct catalytic properties (7–9).

P450 2C9 exhibits a selectivity for the oxidation of relatively small, lipophilic anions such as the non-steroidal anti-inflammatory drugs flurbiprofen, ibuprofen, naproxen, and diclofenac as well as the hypolipidemic agent gemfibrozil. It has been hypothesized that a basic residue in the active site of P450 2C9 stabilizes the binding of these anionic substrates (10–13). However, the structure (Protein Data Bank code 1OG5 [PDB] ) of a chimeric form of P450 2C9 published earlier did not provide evidence for a basic residue that would serve this role. The relatively large size of the active site exhibited by the 1OG5 [PDB] structure rendered the selectivity of P450 2C9 for small lipophilic anions particularly difficult to understand.

Our objective in determining the structure of P450 2C9 was to preserve the native sequence of the enzyme to the greatest extent possible and to co-crystallize the enzyme with a representative anionic substrate bound in the active site to identify structural determinants underlying the substrate selectivity of the enzyme for anionic non-steroidal anti-inflammatory drugs. Eukaryotic P450s are monotopic membrane proteins with the microsomal P450s having a single transmembrane helix at the N terminus that is not found in mitochondrial P450s. Removal of the N-terminal transmembrane domain has facilitated structure determination of the catalytic domains of microsomal P450s (14). However, when expressed in Escherichia coli without their N-terminal transmembrane helices, microsomal P450s generally retain a capacity to bind to membranes (15). In the case of P450 2C5 and the related enzyme P450 2C3, high ionic strength buffers reversed membrane binding (15, 16). A second modification is generally used that extends the C terminus of the enzyme to include a 4-residue histidine tag to facilitate purification of the truncated enzymes. P450 2C9 was modified similarly for crystallization in the present study, and the catalytically active construct is designated P450 2C9dH. In contrast to the modified P450 2C9 employed to determine the 1OG5 [PDB] structure (4), no additional mutations were introduced within the catalytic domain of the protein for the present study.

The structure of the flurbiprofen complex of 2C9dH (Protein Data Bank code 1R9O [PDB] ) reported here differs significantly from the 1OG5 [PDB] structure of the more extensively modified 2C9 construct reported by Williams et al. (4) in specific regions that encompass residues 30–53, 97–121, 196–233, and 467–478. The truncated, histidine-tagged 2C9 construct crystallized in the study of Williams et al. (4) contained seven amino acid substitutions, K206E, I215V, C216Y, S220P, P221A, I223L, and I224L, which correspond to residues in CYP2C5/3LVdH, and the structure, whether bound to warfarin or not, is more similar to that of CYP2C5/3LVdH in this region than the 1R9O [PDB] structure reported here. In addition, warfarin was bound to the 1OG5 [PDB] enzyme in the distal end of the active site cavity with the site of hydroxylation residing in an unproductive position more than 10 Å from the site of oxygen binding, which could reflect the presence of an unidentified substance bound near the iron in the substrate binding site (4). In contrast, the 1R9O [PDB] structure indicates that flurbiprofen is positioned with the site of hydroxylation located for oxidative attack by the reactive, hypervalent iron-oxo intermediate derived from reduction of molecular oxygen by the enzyme. The 1R9O [PDB] structure described in this article also identifies an important role for Arg-108 in stabilizing the binding of flurbiprofen and reveals a distinctly different conformation of the helix B to helix C region, where Arg-108 resides. The 1R9O [PDB] structure contributes significantly to the interpretation of previous mutagenesis studies implicating Arg-108 (17, 18), Asn-289 (7–9), and Asp-293 (19) in substrate recognition and provides a basis for understanding the selectivity exhibited by 2C9 for anionic substrates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
2C9 Construct—The pCW2C9dH construct was prepared by ligating two fragments bearing portions of the CYP2C9 cDNA into a vector derived from the pCW2C5/3LVdH plasmid by digestion with the SacI and SalI restriction enzymes. A SacI-SphI fragment encoding the N-terminal portion of CYP2C9 beginning with codon 23 was excised from the pCW2C9 plasmid described earlier for expression of the full-length enzyme in E. coli (20). The portion encoding the C terminus was PCR-amplified from the plasmid using a lower primer employed previously to incorporate a 4-residue histidine tag at the C terminus of CYP2C5 (16), leading to an alteration of the C-terminal residue of CYP2C9 to a valine plus the histidine tag. The PCR fragment was digested with the restriction endonucleases SphI and SalI, and the three fragments were ligated together to create the expression vector. This strategy fused the coding sequence of 2C9 beginning at residue 23 with the first 5 residues, MAKKT, of the modified N terminus used to express P450 2C5 for crystallization and also replaced the C-terminal valine with an isoleucine plus a 4-histidine tag to facilitate purification (Fig. 1).



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  		FIG. 1.
A, two views depicting the structure of flurbiprofen complexed with 2C9dH. The helices are colored blue, coils gray, and {beta}-sheets brown. The heme is shown as a stick figure, and flurbiprofen is depicted as spheres with carbon, oxygen, nitrogen, and fluorine atoms colored gray, red, blue, and green, respectively. The figure was produced using MOLSCRIPT (36) and RASTER 3D (37). B, the sequence of the 2C9dH construct used to determine the 1R9O [PDB] structure in this study is aligned with the construct used to produce the 1OG5 [PDB] structure. The N terminus of the wild-type enzyme is depicted on the first line. Modifications are shown in bold type in the two truncated constructs. Helices and strands are labeled and indicated by boxed and underlined text, respectively. Residues that are not included in each model exhibit an overstrike.

 
Protein Expression and Purification—P450 2C9dH was expressed and purified as described previously for 2C5dH with several modifications (14). Three l of Terrific broth were seeded with 6 ml of an overnight culture of XL-1 blue E. coli containing the pCW2C9dH plasmid. Following the addition of 240 mg of {delta}-aminolevulinic acid and 720 mg of isopropyl-{beta}-D-thiogalactoside, the culture was incubated at 30 °C for 48 h with shaking at 225 rpm. Cells were harvested by centrifugation and resuspended in 10% of the original culture volume of 20 mM KPi, 20% glycerol, 1 mM phenylmethanesulfonyl fluoride, and 10 mM {beta}-mercaptoethanol. Lysozyme (0.3 mg/ml) was added, and the cells were incubated with stirring for 45 min at 4 °C. An equal volume of cold water was added, and the incubation continued with stirring for an additional 15 min. The spheroplasts were pelleted by centrifugation at 7,000 x g for 10 min, the supernatant was discarded, and the pellets were frozen in liquid nitrogen and stored at –80 °C overnight. The pellet was thawed, resuspended in 500 mM KPi, pH 7.4, 20% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 10 mM {beta}-mercaptoethanol, and sonicated for three 45-s pulses with 1-min cooling on ice between the pulses. Following clarification by centrifugation at 7000 x g for 10 min at 4 °C, CYMAL-5 detergent (Anatrace, Maumee, OH) was added to a final concentration of 4.8 mM, and the material was incubated with stirring at 4 °C for 30 min. Following this incubation, the material was centrifuged at 100,000 x g for 60 min at 4 °C. The supernatant was incubated with 6 ml of nickel-nitrilotriacetic acid resin (Qiagen, Valencia, CA) that was pre-equilibrated with 150 ml of 500 mM KPi, pH 7.4, and 20% glycerol. After 3 h, the resin was collected by gentle centrifugation. The resin was washed with 40 ml of 500 mM KPi, pH 7.4, 20% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 10 mM {beta}-mercaptoethanol, 4.8 mM CYMAL-5 and then with 40 ml of 100 mM KPi, pH 7.4, containing 100 mM NaCl, 20% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 10 mM {beta}-mercaptoethanol, 4.8 mM CYMAL-5. A subsequent wash with 50 ml of 10 mM KPi, pH 7.4, containing 3100 mM NaCl, 20% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 10 mM {beta}-mercaptoethanol, 4.8 mM CYMAL-5, and 1 mM histidine was performed in a small chromatography column. The protein was eluted with 10 mM KPi, pH 7.4, 100 mM NaCl, 20% glycerol, 10 mM {beta}-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 4.8 mM CYMAL-5, and 30 mM histidine. Peak fractions were pooled and diluted with 150 ml of 5 mM KPi, pH 7.4, 20% glycerol, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.2 mM DTT, and 4.8 mM CYMAL-5. The diluted material was then incubated overnight at 4 °C with gentle end-over-end agitation with 5 ml of CM-Sepharose that was equilibrated with the dilution buffer. The following day, the resin was packed in a column and washed with 75 ml of the equilibration buffer containing 1.2 mM CYMAL-5. The P450 eluted sharply in 50 mM KPi, 500 mM NaCl, 20% glycerol, 1 mM EDTA, 0.2 mM DTT, pH 7.4 containing 0.96 mM CYMAL-5, and the pooled fractions contained 165 µM P450. Following elution, the protein was concentrated to 635 µM using a centrifugal membrane-concentrating device with a 50,000 Da cutoff (Millipore, Billerica, MA).

Diclofenac Hydroxylation—P450s 2C9 and 2C9dH (10 pmol) were reconstituted with purified rabbit liver P450 reductase as described previously for P450 2C5dH (15). Reactions were carried out in 1-ml incubations containing 50 mM HEPES, pH 7.4, 0.1 mM diclofenac, and 1 mM NADPH. Following a 30-min incubation, the reaction was terminated by the addition of 0.2 ml of 1 N HCl and placed on ice. The products and substrate were extracted from the reaction mixture and analyzed by reverse-phase chromatography as described previously (21). Elution was monitored by UV absorption at 280 nm, and the integrated elution profiles were compared with those produced by known amounts of 4'-hydroxydiclofenac (BD Biosciences) prepared in the same manner.

Flurbiprofen Binding Studies—The conversion of P450 2C9dH from a low spin to a high spin state in the presence of flurbiprofen was monitored spectrophotometrically as described by Jefcoate (22). Purified protein was diluted to 1.9 µM in 50 mM KPi, pH 7.4, containing 500 mM NaCl, 20% glycerol, 1 mM EDTA, and 0.2 mM DTT in a 1-cm-path length microcuvette. Aliquots of diclofenac dissolved in ethanol were added to the solution, and the differences in the absorption between 394 and 418 nm were recorded. The apparent binding constant was estimated as described previously (8).

Mass Spectral Analysis—Several large crystals were harvested and dissolved in 50 mM KPi, pH 7.4, 500 mM NaCl, 20% glycerol, 1 mM EDTA, and 0.2 mM DTT. The samples were desalted with a ZipTipC4 device (Millipore) according to the manufacturer's protocol and then were analyzed by matrix-assisted laser desorption ionization time-of-flight spectrometry by the Scripps Center for Mass Spectroscopy.

Crystallization and Structure Determination—Hanging drops of 2.5 µl initially containing 0.24 mM P450, 1.92 mM flurbiprofen, 2.3 mM CYMAL-5, 50 mM HEPES, pH 7.5, 1% polyethylene glycol 8000, 20 mM KPi, pH 7.4, 200 mM NaCl, 0.4 mM EDTA, 0.08 mM DTT, and 8% glycerol were equilibrated against 0.1 M HEPES, pH 8.5, and 2% polyethylene glycol 8000 at 24 °C. For x-ray data collection a number of crystals were screened for optimal diffraction. The crystals were prepared by flash-freezing in liquid N2 using a cryoprotectant containing 97 mM HEPES, pH 8.5, 43.5% polyethylene glycol 8000, and 150 mM NaCl. The crystal was then transferred to the cryostream, and data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) beam line 9-1. Data were collected at 100 K using a single crystal and were recorded using a Quantum4 CCD detector and 1° oscillations (180 frames, 90-s exposure). The crystal did not decay noticeably during data collection and exhibited uniform mosaicity of ~0.6°. CCP4 programs MOSFLM and SCALA (23) were used to process the data, and the statistical analysis is presented in Table I.


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  		TABLE I
Data collection and refinement statistics

 
The structure of 2C9 was solved with the 2.3-Å 2C5/3LVdH structure (Protein Data Bank code 1NR6 [PDB] ) as a search model for molecular replacement. Multiple cycles of editing and adjustment of the model into {sigma}A-weighted 2|Fo| – |Fc| composite omit and |Fo| – |Fc| electron density maps were performed using the programs XFIT/XTALVIEW (24) and O (25). The models were refined against the 2.0-Å data using conjugate gradient least squares minimization, simulated annealing, and isotropic individual B-factor refinement using the program CNS (26). During the final stages of refinement, water molecules were added, and flurbiprofen was modeled into the 2|Fo| – |Fc| electron density contoured at 1 {sigma} in the active site of the protein model. Unless otherwise indicated, molecular graphics used in the figures were generated using PyMOL.2

Automated Docking of Dapsone—To examine the potential for simultaneous binding of dapsone with flurbiprofen in the active site of the 1R9O [PDB] structure, computer-based automated docking of dapsone into the active site of the flurbiprofen complex was performed using AUTODOCK 3.05, a grid-based docking program (27). A modified genetic search algorithm employing a local minimum refinement was used to identify low energy binding sites and orientations of the probe molecule. A 50 x 54 x 60-point grid with a spacing of 0.375 Å centered at 2.98, 32.01, –5.25 Å that fully encompassed the active site was used. Explicit polar hydrogens were included in the model. Kollman-united atom charges were assigned to the protein using AUTODOCKTOOLS. Charges for the heme were assigned as described by Helms and Wade (28). Coordinates for dapsone were obtained from the Cambridge Structural Database (CSD). Charges were assigned to flurbiprofen and dapsone using the PRODRG (29) server (davapc1.bioch.dundee.ac.uk/programs/prodrg/). The results of 50 randomly seeded runs were analyzed, and the results were clustered when the atomic coordinates of the final orientation of the docked dapsone molecules exhibited less than a 1-Å root mean square deviation.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The 2C9dH construct was designed to express residues 23–489 of the catalytic domain for crystallization without internal modifications that might affect the structure. The first 22 amino acid residues, which include the transmembrane signal anchor domain, were replaced with the sequence MAKKT (Fig. 1). This sequence was derived from the first 5 residues of the modified N terminus that had been optimized for the expression and crystallization of rabbit P450 2C5 (16). The construct used here differs from that used by Williams et al. (4) to determine the 1OG5 [PDB] and 1OG2 [PDB] structures of CYP2C9 with and without S-warfarin to 2.55 and 2.66 Å, respectively. As shown in Fig. 1, the constructs used for the 1OG5 [PDB] and 1OG2 [PDB] structures incorporated several internal substitutions as well as differences in the N- and C-terminal sequences. Residue numbering for the constructs corresponds throughout the text to that of the wild-type P450 2C9 sequence.

The 1R9O [PDB] construct described here expresses to greater than 400 nmol/liter in E. coli based on whole cell CO-reduced difference spectra and can be extensively purified with only minor modifications to protocols in use for 2C5/3LVdH (14). P450 2C9dH is active when reconstituted with P450 reductase, catalyzing the 4'-hydroxylation of diclofenac, with a kcat value of 11.8 nmol/min/nmol of P450 compared with 14.1 nmol/min/nmol of P450 for the full-length enzyme. Flurbiprofen binds to P450 2C9dH with an apparent binding constant of 9.6 µM determined by monitoring the partial conversion of the enzyme to the high spin ferric form. This value compares closely with a value of 14.1 µM reported for the wild-type enzyme (30). These results suggest that the modifications to the N and C termini do not significantly affect flurbiprofen binding or P450 2C9 oxidation of diclofenac.

Data from a single crystal that diffracted to 2.0 Å in space group R3 with a single molecule in the asymmetric unit were used for model building and refinement. Molecular replacement based on the structure of 2C5 (Protein Data Bank code 1NR6 [PDB] ) was used for initial phasing and as a basis for building the protein model. Subsequent refinement and fitting resulted in a structure with an R value of 0.19 and an Rfree value of 0.24 (Table I). The electron density maps did not define several residues sufficiently for modeling. These include the 7 N-terminal residues of the truncated protein preceding residue 26, residues 38–42 between the proline-rich motif and helix A, residues 214–220 between helices F and G, and the last 2 residues of the C-terminal His tag (Fig. 1). Matrix-assisted laser desorption ionization time-of-flight spectrometry following recovery of the crystallized protein indicated an atomic mass of 54,160 Da (with a predicted atomic mass of 54,151 Da), confirming that the crystallized protein had not been cleaved. Additionally, the final model included 1 glycerol and 255 H2O molecules (Table I). The 2|Fo| – |Fc| electron density maps contoured at 1.0 {sigma} clearly defined the position and orientation of flurbiprofen in the active site, which was modeled as S-flurbiprofen based on electron density defining this diastereomer. The experimentally determined structure of 2C9dH displays good stereochemistry with only 1.6% of the residues found in generously allowed or disallowed regions of the Ramachandran plot (Table I).

The 1R9O [PDB] structure of the flurbiprofen complex of 2C9dH corresponds closely to the 1OG5 [PDB] structure in many regions with a root mean square deviation of 0.74 Å. However, significant differences are seen in the N terminus preceding residue 53 in helix A, residues 97–121 between helices B and C, residues 197–232 encompassing helix F through the beginning of helix G, and residues 467–478 that form the turn in the C-terminal antiparallel {beta}-sheet (Fig. 1). These differences are clustered in one quadrant of the structure (Fig. 2), which also contains regions that are poorly ordered and could not be modeled precisely. These regions of the structure exhibit conformational flexibility in other P450s (5, 31). The 1R9O [PDB] structure exhibits an extra turn at the beginning of helix A that is not seen in other mammalian P450 structures including 1OG5 [PDB] . The packing of the extended helix A with {beta}-sheet 4 displaces the latter from the position seen in the 1OG5 [PDB] structure.



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  		FIG. 2.
Comparison of the 1R9O [PDB] structure (cyan) with the 1OG5 [PDB] structure (gray). Ribbons depict the C{alpha}-trace of the two structures near the N-terminal end of helix A. Helices F' and G' are not evident in the 1R9O [PDB] structure, which exhibits a more extended conformation of the region between helices F and G and a longer helix A. A divergent stereo view is shown.

 
Helix G' is not evident in the 1R9O [PDB] structure. The corresponding region forms an extended structure with attendant shifts in the conformation of residues that would correspond to helices A' and F'. In this regard, it is important to note that the sequence of the 1OG5 [PDB] sequence differs from the native enzyme in the helix G' region (Fig. 1). The substitutions introduced into the helix F–G region of the 1OG5 [PDB] construct correspond to the sequence of the 2C5 construct crystallized in earlier studies from our laboratory (15). Most notably, the S220P and P221A mutations in 1OG5 [PDB] move Pro-221 to a position seen for Pro-220 in 2C8 and 2C5, where it forms a tight turn between helices F' and G'. The repositioning of the proline may stabilize the helix F'–G' region of P450 2C9 in a conformation similar to that seen in P450s 2C8 and 2C5. In addition, the P221A, L222I, and L223I substitutions could increase the propensity of this region to form helical structures. The distinct conformation of the helix G' region exhibited by the native sequence in the 1R9O [PDB] structure is consistent with previous studies of chimeric enzymes constructed from 2C9 and 2C19, which have highly similar amino acid sequences. Analysis of chimeras indicated that the S220P and P221S differences between the two enzymes contribute to the distinct selectivity of 2C19 for the substrates omeprazole (32) and S-mephenytoin (7). However, it is not necessary to introduce the reverse mutations into 2C19 to confer diclofenac hydroxylation (9) or sulfaphenazole binding (8) to 2C19. Thus, the difference in the conformation of this region between the 1OG5 [PDB] and 1R9O [PDB] structures may not contribute to the substrate selectivity of 2C9 for small lipophilic anions.

The 2C9dH crystals were grown in the presence of an 8-fold molar excess of racemic flurbiprofen. The location of flurbiprofen is well defined in the active site by the electron density map phased by the protein model without the substrate (Fig. 3). Details of the map indicate the location of the fluorine substituent and the orientation of the carboxylate. An additional, weaker density consistent with the S-enantiomer suggests a preference for binding S-flurbiprofen, which is consistent with experimental observations. However, this does not preclude partial, lower occupancy by the other diastereomer that might not produce significant electron density. R-Flurbiprofen has been reported to exhibit only slightly lower Km values for oxidation by 2C9 (33). The carboxylate side chain of flurbiprofen forms a salt bridge with Arg-108 and hydrogen bonds to Asn-204. Arg-108 in the 1OG5 [PDB] structure of 2C9 (Fig. 4) as well as Lys-108 in the structures of 2C5 (Protein Data Bank code 1NR6 [PDB] ) and 2C8 (Protein Data Bank code 1PQ2 [PDB] ) are oriented away from the active site cavity. In the flurbiprofen complex of 2C9, residues in the helix B to helix C region adopt a distinctly different conformation allowing Arg-108 to interact with the negatively charged substrate and residues Asn-289 and Asp-293 on helix I (Fig. 4). This region exhibits significant flexibility in the structures of 2C5 (5) and 2B4 (31) and can adopt alternative conformations without greatly altering the secondary structure immediately before and after because of the presence of glycine residues that flank residues 97 and 110 (Fig. 1). These GXG motifs flanking the flexible region can serve as swivel points for the encompassed segment, which allow localized conformation changes without significantly perturbing other portions of the structure.



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  		FIG. 3.
2|Fo| – |Fc| electron density map contoured at 1.0 {sigma} and all data to 2.0-Å resolution. The electron density map was generated with the substrate omitted from the model. A divergent stereo view is shown. For clarity, the map was not rendered for other portions of the model. The heme is colored salmon, and the atoms of the protein and substrate are colored gray for carbon, red for oxygen, blue for nitrogen, and magenta for fluorine.

 



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  		FIG. 4.
Stereo views of residues 97–114 in the 1R9O [PDB] and 1OG5 [PDB] structures. The ends of each sequence match closely, but the intervening regions exhibit different conformations that radically alter the position of the side chain of Arg-108. In addition, flurbiprofen binds close to the heme in the 1R9O [PDB] structure, whereas warfarin resides further away and on the other side of Phe-114. Different locations of Phe-114 and Phe-100 are required to accommodate warfarin in this position. A divergent stereo view is shown.

 
Hydrogen bonding of Arg-108 with two residues on helix I, Asp-293 and Thr-289, stabilizes the observed conformation of Arg-108 in the active site (Fig. 3). This result indicates that Arg-108 is likely to be critical for the relatively high affinity of flurbiprofen binding to 2C9. Substitution of other amino acids for Arg-108 has been shown to alter flurbiprofen binding (18) and to diminish greatly the oxidation of S-warfarin (18) and diclofenac (17, 18). Interestingly, hydroxylation of pyrene, a neutral substrate that is not capable of hydrogen bonding with Arg-108, was largely unaffected by the R108F substitution (18). Mutagenesis of Asp-293 (18, 19) also profoundly affects the oxidation of diclofenac, another anionic substrate, by 2C9. However, this mutation also affects the stability of the enzyme (18). Asp-293 exhibits hydrogen bonding interactions that are likely to stabilize the structurally adjacent turn formed by the polypeptide backbone at residues 111–114 (Fig. 4). The observation that Asn-289 of 2C9 provides additional hydrogen bonding interactions that stabilize the conformation of Arg-108 in the active site cavity is consistent with the observation that the I289N substitution of the closely related enzyme 2C19 confers diclofenac oxidation to 2C19 (9) as well as high affinity binding of the selective 2C9 inhibitor sulfaphenazole (8). Thus, the 1R9O [PDB] structure provides a rationale for the effects observed in several mutagenesis studies.

The substrate is positioned so that the site of hydroxylation resides 4.9 Å from the heme iron, which is consistent with the distances seen in other P450 substrate complexes. A water molecule, W600, resides between flurbiprofen and the heme iron (Fig. 5). The water is 2.4 Å above the iron and 3.0 Å from flurbiprofen. The water molecule, 600, is ordered by its interactions with the heme iron and potential hydrogen bonding interactions with the backbone carbonyl of Ala-297. Flurbiprofen produces only a partial increase in high spin character when it binds to the enzyme, which is consistent with partial occupancy by water of the sixth coordination site of the iron in the predominantly low spin heme protein. A second water molecule, 819, is evident in the distortion of helix I as it passes over the heme prosthetic group as seen in many other P450 structures.



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  		FIG. 5.
Amino acid side chains residing within 5 Å of flurbiprofen. Amino acid side chains, a glycerol, and flurbiprofen are rendered as stick figures with carbons colored white except in flurbiprofen, where carbons are colored cyan. Oxygen, nitrogen, sulfur, and fluorine are colored red, blue, yellow, and purple, respectively. For reference, the heme is shown as a stick figure colored salmon, and a portion of helix I is rendered as a ribbon with the position of Gly-296 shown. Water molecules 600, 819, and 842 are rendered as red spheres. A divergent stereo view is shown.

 
Flurbiprofen packs next to helix I in a hydrophobic cleft that is formed by Val-113 and Phe-114 in the SRS 1 region; Ile-205 and Leu-208 in SRS 2; Val-237 and Met-240 in SRS 3; Val-292, Gly-296, and Ala-297 in SRS 4; and Leu-366 in SRS 5 (Fig. 5). The cleft surrounds flurbiprofen on three sides but leaves flurbiprofen exposed to solvent in the larger active site cavity. A water molecule, 842, and a glycerol reside between the substrate and the turn in the C-terminal {beta}-sheet (SRS 6) (Fig. 5). The solvent channel between helix F, helix I, and the turn in the C-terminal antiparallel {beta}-sheet motif is relatively open. The absence of direct contacts from SRS 6 and the N-terminal portion of SRS 1 is notable and reflects the relatively large active site cavity when compared with the size of flurbiprofen. This cavity is partially occupied by glycerol and two water molecules with defined density.

Dapsone has been reported to stimulate the oxidation of flurbiprofen by P450 2C9, and kinetic analyses are consistent with a model where both dapsone and flurbiprofen occupy the active site cavity (34). The relatively large active site cavity compared with the size of flurbiprofen (Fig. 6) suggested that there might be sufficient space to accommodate dapsone together with flurbiprofen in the 1R9O [PDB] structure. To test this possibility, automated docking studies employing AUTODOCK 3.05 (27) were used to examine the potential fit of dapsone in the structure of the flurbiprofen complex. Fifty randomly seeded searches using a genetic algorithm combined with refinement of local minima identified three potential binding locations. The solution displaying the lowest predicted binding energy is depicted in Fig. 6. The predicted location of the dapsone molecule is under helix F between flurbiprofen and the C-terminal {beta}-sheet (SRS 6). Although the cavity appears to be closed, the surface was terminated by external water molecules at each end. The docking result suggests that binding of dapsone in this position might serve to limit the motion of flurbiprofen in the active site so that it remains closely positioned for oxidation; furthermore, this binding may displace solvent molecules and shield the perferryl oxo intermediate from hydrogen ion transfer from the bulk solvent. These mechanisms could contribute to the observed increase of catalytic efficiency for the oxidation of flurbiprofen in the presence of dapsone. However, confirmation of this speculation will require more extensive experimental investigation.



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  		FIG. 6.
Solvent-accessible surface of the substrate binding cavity of CYP2C9. The surface is represented by a fine mesh and was calculated using a 1.4-Å probe with the program VOIDOO (38). Portions of the protein bounding the cavity are rendered as a ribbon, and the heme is rendered as a stick figure colored salmon. Carbon atoms are colored green, cyan, and white for flurbiprofen, dapsone, and the protein, respectively. Oxygen, nitrogen, sulfur, and fluorine are colored red, blue, yellow, and brown, respectively. A divergent stereo view is shown.

 
In summary, the structure of the P450 2C9-flurbiprofen complex provides new insights into substrate binding to the enzyme. The 1R9O [PDB] structure is consistent with several experimental observations that were difficult to rationalize based on the earlier 1OG5 [PDB] structure. The 1R9O [PDB] structure reveals that Arg-108 plays a significant role in the binding of flurbiprofen to the enzyme, and it is likely to contribute to binding of other lipophilic anions that are substrates for P450 2C9 such as naproxen, ibuprofen, diclofenac, indomethacin, and gemfibrozil. The conformation of Arg-108 seen in the 1R9O [PDB] structure is stabilized by interactions with two residues on helix I, Asp-293 and Asn-289. Although both Arg-108 and Asp-293 are conserved in P450 2C19, Asn-289 is not, and substitution of Asn at this position in P450 2C19 confers diclofenac hydroxylation to the enzyme (9). The conformation of the helix B to helix C region (SRS 1) observed in the 1R9O [PDB] structure differs significantly from the 1OG5 [PDB] structure, which is more typical of conformations observed in structures of 2C5 (Protein Data Bank codes 1NR6 [PDB] and 1N6B [PDB] ), 2C8 (Protein Data Bank code 1PQ2 [PDB] ), and 2B4 (Protein Data Bank code 1SUO). The structures of the open and closed forms (Protein Data Bank codes 1PO5 [PDB] and 1SUO, respectively) of P450 2B4 indicate extensive conformational changes that the helix B to helix C and helix F to helix G regions can adopt and that are likely to occur in solution (31, 35). Although the sequences of the two P450 2C9 constructs used for crystallization differ, the differences evident between the 1R9O [PDB] and 1OG5 [PDB] structures are likely to reflect the conformational flexibility of the protein. Determination of the role of conformational changes in the binding of individual substrates by drug-metabolizing P450s as well as the relative population of stable open and closed states of P450 enzymes presents challenging but important questions for understanding the structural determinants of substrate oxidation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM031001 (to E. F. J.). Facilities for computer-assisted sequence analysis, DNA sequencing, and the synthesis of oligonucleotides were supported in part by General Clinical Research Center Grant M01 RR00833 and by the Sam and Rose Stein Charitable Trust. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the United States Department of Energy, Office of Biological and Environmental Research and by the National Center for Research Resources, Biomedical Technology Program, and NIGMS of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1R9O [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence may be addressed: Dept. of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB8, La Jolla, CA 92037. Tel.: 858-784-8738; Fax: 858-784-2857; E-mail: dave{at}scripps.edu. ** To whom correspondence may be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MEM-255, La Jolla, CA 92037. Tel.: 858-784-7918; Fax: 858-784-7978; E-mail: johnson{at}scripps.edu.

1 The abbreviations used are: CYP or P450, a generic term for a cytochrome P450, with individual P450s identified using a number-letter-number format based on sequence relatedness; KPi, potassium phosphate; DTT, dithiothreitol; Fo, observed structure factor; Fc, calculated structure factor; SRS, substrate recognition site. Back

2 W. L. DeLano, PyMOL Molecular Graphics System (2002) at www.pymol.org. Back



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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