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Originally published In Press as doi:10.1074/jbc.M312516200 on December 15, 2003

J. Biol. Chem., Vol. 279, Issue 10, 9497-9503, March 5, 2004
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Structure of Human Microsomal Cytochrome P450 2C8

EVIDENCE FOR A PERIPHERAL FATTY ACID BINDING SITE*

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

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

Received for publication, November 16, 2003 , and in revised form, December 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A 2.7-Å molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the {beta}1 sheet and helix F' that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F', G', and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Xenobiotic metabolizing cytochrome P450 monooxygenases provide crucial protection from the harmful effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. In general, these microsomal enzymes determine the bioavailability of hydrophobic compounds by controlling the rate of conversion to more soluble, inactive products that are readily excreted. Different P450s can show overlapping substrate specificities, and individual enzymes can interact with numerous structurally diverse substrates. This broad catalytic activity usually serves a positive defensive role; however, in some cases, it can lead to adverse drug-drug interactions.

P450s often possess enzyme-specific catalytic repertoires and can display exquisite catalytic selectivity for regio- and stereospecific reactions. This is particularly evident for the mammalian family 2C P450s, which exhibit extensive, independent evolution and functional divergence in mammals, leading to multiple enzymes in each species while retaining a high degree of amino acid identity (>70%). Thus, structural comparisons within this highly related but functionally diverse P450 subfamily are likely to be particularly revealing of mechanisms leading to the catalytic diversity of P450 enzymes.

The structural features of drug-metabolizing P450s that contribute to their capacity to oxidize structurally diverse substrates while maintaining site-specific oxidation is an active area of research because of the obvious benefits that this information could provide regarding xenobiotic risk assessment, the prediction and avoidance of negative drug-drug interactions, as well as the rational design of improved therapeutic drugs and specific inhibitors. Herein, we report the first structure of a catalytically active, human microsomal cytochrome P450 2C8 (CYP2C8)1 obtained without the use of multiple internal mutations to the catalytic domain. Analysis of this structure suggests several general mechanisms for altering the topography and function of the P450 active site. These include residue substitutions that affect the volume available for substrates, regional flexibility to enhance substrate interactions, and peripheral binding sites that can modulate active site characteristics and enable dimerization to further restrict localized adaptive changes that can influence substrate binding and oxidation.

Human CYP2C8 plays a central role in the metabolism of a number of therapeutic drugs. This enzyme is expressed at relatively high levels in the liver (1, 2), which is often the principal site for drug clearance. CYP2C8 has been shown to contribute extensively to the clearance of the anticancer drug taxol (3, 4), the antimalarial drug amodiaquine (5), the antidiabetic drugs troglitazone (6) and rosiglitazone (7), the anti-arrhythmic drug amiodarone (8), and the calcium channel blocker verapamil (9). CYP2C8 is the primary enzyme that metabolizes a cholesterol lowering drug cerivastatin, and inhibition of its metabolism by a CYP2C8 inhibitor, gemfibrozil, a lipid lowering drug, causes a toxic drug-drug interaction that provokes rhabdomyolysis (10-12). CYP2C8 is also involved in the metabolism of natural substrates like unsaturated fatty acids and retinoic acid (13). In human liver and kidney, CYP2C8 is the predominant P450 responsible for the oxidation of arachidonic acid to generate biologically active epoxyecosatrienoic acids that are involved in the regulation of blood pressure (14, 15).

The ability to determine structures for the human drug-metabolizing P450s has been hindered by the inherent difficulties associated with crystallizing membrane enzymes. Recently, our laboratory engineered rabbit microsomal CYP2C5 for expression in Escherichia coli as a conditionally soluble membrane protein that retained catalytic activity (16). Crystallization of the protein led to the first structure of a microsomal P450 (17). Similar modifications to the N terminus allowed us to express the native sequence of the CYP2C8 catalytic domain for structural studies. The experimentally determined structure diverges significantly from the structure of CYP2C5 in ways that are critical for function. The major structural differences contribute to a much larger substrate binding site cavity in CYP2C8 that is compatible with its capacity to oxidize relatively large substrates such as taxol. Surprisingly, CYP2C8 crystallized as a symmetric dimer formed by the interaction of the helix F to G regions of the protein. Two molecules of palmitic acid are bound in the dimer interface. This novel finding suggests the existence of a peripheral binding site that can affect the structural dynamics of the active site and may underlie the cooperative effects of drug-drug interactions in P450 metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction and Purification of CYP2C8dH—The CYP2C8 cDNA used for modifications was kindly provided by Robert Tukey (University of California, San Diego). The CYP2C8 cDNA was modified by replacing the sequence encoding the first 27 amino acids with one encoding a short hydrophilic, positively charged N terminus (MAKKTSSKG) identical to that employed for the expression and crystallization of CYP2C5 (16, 18). This modification of the N terminus of CYP2C8dH converts the integral membrane protein to one that binds peripherally to membranes and that can be separated from the membrane by elevating the ionic strength of the medium. In addition, a four-residue histidine tag was added to the C terminus to facilitate purification. The modified cDNA was cloned into the pCWori plasmid for expression in E. coli. The resulting enzyme is designated CYP2C8dH.

The modified enzyme was isolated in the presence of the detergent CYMAL5 (Anatrace, Maumee) as described for CYP2C5dH (19) with some modifications. Briefly, CYP2C8dH was purified from E. coli lysates by metal ion affinity column chromatography (nickel-nitrilotriacetic acid, Qiagen Inc., Valencia, CA). Following elution of the protein in a buffer containing 40 mM histidine, the protein was subjected to CM-Sepharose ion exchange chromatography (CL6-B, Amersham Biosciences, Piscataway, NJ) to deplete the detergent. The purified protein was concentrated using a centrifugal device (Ultrafree-15 50K, Millipore, Billerica). CYP2B4dH and CYP2C9dH constructs were expressed and purified in a similar way as CYP2C8dH. The expression vector for 2B4dH (20) was kindly provided by Emily Scott and Jim Halpert (University of Texas Medical Branch).

Characterization of CYP2C8dH—The purified protein exhibited a specific P450 content of 18 nmol/mg of protein, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein indicated a molecular mass of 53,988 Da, which agrees with the 53,979 Da predicted from the amino acid composition of CYP2C8dH. The concentration of P450 was determined as described previously (21). Protein concentrations were determined using bicinchoninic acid reagent (Pierce Biotechnology, Rockford, IL).

Based on an analysis of type I difference spectra (22), purified CYP2C8dH binds arachidonic acid (Sigma-Aldrich, St. Louis, MO) with an apparent KD of 1.8 µM with a maximum 27% conversion of the enzyme to the high spin form. This binding constant is 6-fold lower than values measured previously for the partially purified wild type enzyme (14). Taxol also produced a partial conversion of the enzyme to the high spin form, but the limited solubility of this substrate precluded an estimate of the binding affinity. The rate of 6{alpha}-hydroxylation of taxol (10 µM) was determined to be 0.5 nmol/min/nmol of P450, when CYP2C8dH (10 pmol) was reconstituted with 0.28 unit (1 µmol of cytochrome c reduced/min/mg) of P450 reductase in 50 mM HEPES, pH 7.5, in the presence of 30 µg of dilaurylphosphatidylcholine in a total volume of 1 ml at 37 °C for 15 min. Product formation was analyzed by high-performance liquid chromatography as described (23) using 6{alpha}-hydroxytaxol (Gentest) as a quantitative standard. The substrate was not soluble at higher concentrations precluding a determination of Vmax.

Crystallization and Data Collection—The protein was crystallized by the vapor diffusion method using 2.5-µl sitting drops containing 1.25 µl of 392 µM P450, 1.12 mM CYMAL-6 detergent (Anatrace, Maumee), 40 mM potassium phosphate, pH 7.4, 400 mM NaCl, 0.8 mM EDTA, 0.16 mM dithiothreitol, and 16% glycerol and 1.25 µl of 100 mM HEPES, pH 7.5, 15% ethanol, and 10% PEG4000. The drops were equilibrated with 100 mM HEPES, pH 7.5, 15% ethanol, and 10% PEG4000 at 24 °C. For data collection, the crystal was soaked for 2 min in a cryoprotectant composed of 70 mM HEPES, pH 7.5, 10.5% ethanol, 7% PEG4000, and 30% ethylene glycol, and then flash-frozen in liquid N2 prior to transfer into the cryo-stream for data collection at Stanford Synchrotron Radiation Laboratory beam line 7-1. The data were collected at 100 K for a single crystal of dimensions 0.15 x 0.15 x 0.4 mm using a MAR345 image plate (Mar Research) with 2° oscillations (50 frames, 300-s exposure). The crystal did not decay noticeably during data collection. The data were processed with CCP4 programs MOSFLM and SCALA (24, 25). CNS (version 1.1) (26) was used for initial phasing by molecular replacement and subsequent refinement protocols. Xfit/Xtalview (27) and O (28) were employed for display electron density maps and model building.

Structure Determination—The structure of CYP2C5/3LVdH (PDB ID code 1N6B [PDB] ) was used as the template for model building via molecular replacement. Divergent residues were truncated to C{beta} in the search model. Molecular replacement was carried out with CNS (version 1.1) (26). Standard refinement protocols were employed, and the refinement proceeded normally through several cycles of interpretation, editing, and adjustment of the model into {sigma}A-weighted 2 Fo - Fc and Fo - Fc (where Fo = observed structure factor and Fc = calculated structure factor) electron density maps using either Xfit/Xtalview (27) or O (28). Following rebuilding, subsequent models were refined, using CNS, by simulated annealing (using non-crystallographic symmetry restraints in the first rounds), and individual atomic, isotropic B-factor refinement. In addition to small individual adjustments of backbone and side chain, the peptide backbone for residues 207-217 (F/G loop) and 271-277 (polar surface loop) were entirely rebuilt, and one palmitic acid was added per protein molecule in the asymmetric unit and adjusted into the observed density.

Extraction and Characterization of Fatty Acids Associated with CYP2C8dH—Tetradeuterated palmitic acid (5 nmol) (Medical Isotopes) was added to 10 nmol of different preparations of purified P450s for use as an internal standard. After an incubation with 0.5 nmol of proteinase K, 1 volume of 10 mM NaOH was added to facilitate the release of bound fatty acid. After a second addition of 1 volume of 100 mM NaOH, the pH was decreased with an excess of HCl. Samples were extracted with 2.5 volumes of organic phase (ether/hexane, 50/50), dried under nitrogen, reconstituted in methanol, and analyzed by electrospray ionization mass spectrometry (PerkinElmer Life Sciences API 100 Sciex single quadrupole).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Structure Determination—The structure of CYP2C8dH was determined from data collected for a single crystal diffracting to 2.7 Å. The enzyme crystallized in the C2 space group with two molecules per asymmetric unit. The structure was solved initially by molecular replacement using the structure of CYP2C5/3LVdH (PDB ID code 1N6B [PDB] ) (29). The initial maps were highly interpretable, and the overall structure is very similar to that of CYP2C5/3LVdH. However, additional refinement and rebuilding were necessary to construct divergent regions. This included portions of the polypeptide backbone that form the active site cavity. The final models for both molecules in the asymmetric unit exhibited connective electron density for residues 28-490, where the numbering corresponds to that of the native enzyme, with an R-factor of 24.7% (Rfree 28.4%) (Table I). Backbone geometry was analyzed in PROCHECK (30) and only one residue (Gln278 in molecule B) was in the disallowed region of the Ramachandran plot. The two molecules of CYP2C8dH in the asymmetric unit are highly similar with an root mean square deviation of 0.52 Å (for 461 C{alpha} atoms) and average B values of 60.2 Å2 and 65.8 Å2, respectively, for the A and B molecules.


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  		TABLE I
Data collection and refinement statistics

 
Crystallization of CYP2C8dH as a Dimer Stabilized by Fatty Acids Bound in the Interface—The two molecules in the asymmetric unit form a symmetric dimer with the regions between helices F to G of molecules A and B interacting extensively (Fig. 1A). CYP2C8dH exhibits the typical P450 fold illustrated in Fig. 1B. Following the completion and building of the protein model, additional electron density was present within the dimer interface (Fig. 1C) that suggested the presence of two fatty acid molecules. A palmitic acid molecule, a C16 saturated fatty acid, was built into a long, almost continuous, tubular density with a branched terminus that resides near the N-terminal end of helix G of the dimerization partner. The average B values are slightly lower for the two palmitic acids (60.5 Å2) than the average values exhibited by the protein molecules (63.2 Å2) (Table I).



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  		FIG. 1.
Structure of CYP2C8 and the fatty acid binding site at the dimerization interface. A, stereo view of the CYP2C8dH dimer in the asymmetric unit. Two palmitic acid molecules bound at the interface stabilize the dimer. The palmitate molecules are rendered as using a space filling atom representation with the carbons colored gray. The heme is represented in gray. Molecule A of the dimer is on the top. The peptide chains of both P450s are colored by temperature factors ranging from 45 to 85 Å2 (rainbow from blue to red). Unlike other P450 structures, the region between F and G helix does not exhibit the highest temperature factors. The N-terminal anchor domains of both molecules are positioned on the same side of the homodimer indicating an orientation compatible with membrane binding. The proximal surfaces of each P450 are roughly parallel and would be accessible for interaction with P450 reductase bound to the membrane. The figure was produced in Molscript (41) and Raster 3D (42). B, view of the distal face of CYP2C8dH with {beta}-sheets depicted in tan, loops in gray, and helices in green. The helices and {beta}-sheets are labeled sequentially from the N terminus according to common usage for P450 structures. The figure was produced in Molscript and Raster 3D. C, stereo view of the fatty acid binding sites at the dimer interface. The sigma-A-weighted composite omit maps (gray) show the electron density peaks that were modeled with the two palmitate molecules yielding correlation coefficients of 0.90 and 0.875, and real space R-factors of 10% and 12.5% for the A and B models, respectively. Residues from molecule A in the dimer are depicted in yellow, and side chains from molecule B are shown in green. Hydrophobic residues surround the aliphatic chain. The carboxyl groups of the palmitate molecules fit the Y shape of the electron density and interact with the first turn of helix G in the dimerization partner. The non-crystallographic 2-fold axis passes between residues 226 and 229. The figure was generated using Xfit (27) and Raster 3D.

 
The terminal portion of the aliphatic chain of the palmitic acid resides in a tubular, hydrophobic cavity formed by helices F', G', and G of each protein molecule (Fig. 1A). The carboxylate group of the fatty acid interacts with the backbone amides in the first turn of helix G of the second molecule in the dimer (Fig. 1C). Thus, the binding of the ionized fatty acid is likely to be stabilized by the helix dipole as well as by direct hydrogen bonding interactions with the backbone amides forming the first turn of helix G. A portion of the aliphatic chains of the two fatty acids are exposed to each other and are separated by roughly 5 Å, which adds additional hydrophobic surface area to the interaction at the dimer interface.

Size exclusion chromatography indicates that the isolated protein is predominantly a dimer in solution with evidence for a small amount of monomer (Fig. 2). The dimer tends to dissociate at low P450 concentrations. Ether/hexane extracts of the enzyme following proteolytic digestion and neutralization were characterized by electrospray ionization mass spectrometry (Fig. 3). The chromatograms revealed a molecular ion with a mass that is consistent with the presence of palmitic acid, m/z 255. A second, prominent molecular ion, at m/z 283, could represent the C18 fatty acid, stearic acid. These ions were not observed in extracts from other P450s that were expressed and purified in a similar manner. It is likely that the fatty acid molecules were acquired in the E. coli host used for expression of the protein, and palmitic and stearic acid are the predominant saturated fatty acids in E. coli. The density observed for the fatty acid (Fig. 1C) is consistent with the 16 carbon aliphatic chain of palmitic acid. However, partial occupation of the site by stearic acid cannot be excluded.



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  		FIG. 2.
CYP2C8dH is a dimer in solution. 10 nmol of purified CYP2C8dH (in 250 µl) were applied to a Superdex 200 column (HR 10/30, Amersham Biosciences) as described previously (16). Comparison to the standard curve (lower panel) indicates apparent molecular masses of 113 kDa for the dimer, and 38 kDa for the monomer. The predicted molecular mass of the dimer is 108 kDa. The molecular mass standards used were {beta}-amylase (200 kDa, 25.3 min), alcohol dehydrogenase (150 kDa, 26.8 min), bovine albumin (66 kDa, 29.3 min), ovalbumin (45 kDa, 31.6 min), and cytochrome c (12.4 kDa, 36.9 min). Protein elution was monitored by measuring the absorbance of the eluate at 280 or 417 nm with an inline detector.

 



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  		FIG. 3.
Mass spectral evidence for bound fatty acid. Negative ion electrospray mass spectra of ether/hexane extracts from 10 nmol of CYP2C8dH and CYP2C9dH are shown. Tetradeuterated-palmitic acid (5 nmol, peak 259 Da) was added to the proteins prior to extraction to provide an internal standard. Two peaks that correspond to m/z values for palmitate (255 Da) and stearate (283 Da) are present in the CYP2C8dH extract. These are not observed in extracts from CYP2C9dH, CYP2B4dH, or buffer-only samples (not shown). The molar ratio of bound fatty acid from three different purified CYP2C8dH preparations ranges from 35 to 77%, with a constant molar ratio of palmitic/stearic acid of 1.9 ± 0.1.

 
Comparison of the 2C8 and 2C5 Active Sites—Examination of the structure of CYP2C8dH indicates that the active site cavity is roughly twice as large as that of CYP2C5/3LVdH (Fig. 4). This is consistent with the capacity of CYP2C8 to oxidize larger substrates such as taxol. The increased volume of the active site results not only from changes in the conformation of portions of the protein that include helices F' and B', which delimit the distal portion of the active site cavity, but also from differences in the volume of side chains that occupy the cavity. The smaller volume of the CYP2C5 active site largely reflects the presence of two juxtaposed phenylalanine residues, Phe114 and Phe473 (Fig. 4). In CYP2C5, Leu213 of helix F' is packed behind these two residues, and together they partition that section of the binding site cavity in CYP2C5. In CYP2C8, the corresponding residues are smaller, Ser114 and Ile476, and the distance between side chains increases from 5.6 Å (closest atom center to closest atom center) in CYP2C5 to 13.5 Å in CYP2C8. The size of the active site cavity is expanded through the opening between residues 114 and 476, because helix F' of CYP2C8 is positioned up and away from these two residues, which results in a significantly larger active site cavity. This reflects a 9-Å difference in the position of the Trp212 C{alpha} atom at the corner between helices F and F' (Fig. 4C). Additional factors that contribute to the larger active site volume of CYP2C8 are the altered position of the B' helix, which differs by roughly 2.4 Å from CYP2C5, and the side chains of the residues on the B'-helix that project into the substrate binding cavity are smaller in CYP2C8 than the corresponding residues in CYP2C5.



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  		FIG. 4.
Comparison of the active site cavities and F'-G' regions in P450s 2C8 and 2C5. Solvent-accessible surfaces of the active site cavities of CYP2C8dH (A) and CYP2C5/3LVdH (B) were computed using the program VOIDOO (43) and are represented as a mesh. The active site volume of CYP2C8dH is estimated to be 1438 Å3, whereas the active site volume of CYP2C5/3LVdH (PDB ID code 1N6B [PDB] ) is estimated to be 645 Å3. A solvent access channel indicated by an arrow is apparent for CYP2C8dH. C, the backbones of the F'-G' region of CYP2C8 (depicted in green) and CYP2C5 (depicted in yellow) are overlaid and displayed as ribbons. In CYP2C8, residues 208-216 form the F' helix. In CYP2C5, residues 211-213 form a sharp bend that extends the conformation toward {beta}-sheet 1. The relative distance between the C{alpha} atoms of residue Trp-212 in the two structures is 9 Å. In CYP2C8, a palmitate molecule is bound in the F'-G' region. The heme is depicted in red. The figure was generated in PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System (2002), available at www.pymol.org).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The structure of CYP2C8 reported here illustrates several structural features of the xenobiotic metabolizing P450s that reflect the rapid genetic and functional diversification of these enzymes between mammalian species. Although the mammalian CYP2C enzymes generally exhibit greater than 70% amino acid identity and a higher degree of amino acid similarity, the genes encoding these enzymes exhibit extensive, independent duplication and diversification in mammalian species. As a consequence, the number of CYP2C enzymes varies among species, and each enzyme exhibits a distinct spectrum of substrate and inhibitor selectivity. This appears to reflect a high rate of non-synonymous nucleotide substitutions that alter amino acids that form the substrate binding site and contribute to the capacity to oxidize and detoxify a wide range of xenobiotics (31). Of the 25 amino acid side chains that are in the active sites of both CYP2C8 and CYP2C5, the side chains differ for 18 (Table II).


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  		TABLE II
Active site residues in P450s 2C8 and 2C5

The numbering of the residues is based on the full-length enzymes. The active site of P450 2C8 is much larger than that of 2C5 and reveals 23 additional residue positions that potentially contact substrates (boldface). Conserved residues that reside in the active sites of both enzymes are underlined. A three-residue insertion at position 275 in 2C8 shifts the corresponding number relative to P450 2C5.

 
A comparison with the structure of CYP2C5 (29) (PDB ID code 1N6B [PDB] ) indicates that the active site volume of CYP2C8 is roughly twice as large as that of CYP2C5. This reflects both conformational changes in the polypeptide backbone and the size of amino acid side chains that reside in the substrate-binding cavity. There are 23 more amino acid side chains in the active site of CYP2C8 when compared with CYP2C5. This results from the inclusion of amino acid residues from additional portions of the structure in the active site cavity such as {beta} sheet 1 and helix F'. Of the 18 differences in amino acid side chains that are found in the active site cavities of both enzymes, 10 exhibit significant differences in side-chain volume and/or polarity (Table II). For example, CYP2C8 has a cluster of small polar residues on the active site side of the B' helix (Ser100, Ser103, and Ser114), where larger hydrophobic residues are found in CYP2C5.

The increased size of the CYP2C8 active site when compared with CYP2C5 also reflects a difference in the position of helix F' that creates a cavity above {beta} sheet 1. The occurrence of small side chains for the residues Ser114 and Ile476 in CYP2C8 allows the active site cavity to extend farther from the heme and incorporate the additional space afforded by the conformation of the F' helix (Fig. 4). The larger size of the active site cavity is consistent with the observation that CYP2C8 can efficiently oxidize relatively large substrates such as paclitaxel, cerivastatin, amiodarone, and amodiaquine, which are not good substrates for other human CYP2C enzymes.

Other P450 structures indicate that the helix F to G region is flexible and can adopt different conformations in structures of the same enzyme with different substrates. Thus, substrate binding could alter the position of helix F' and as a consequence affect the volume of the active site cavity. This region exhibits different conformations in CYP2C5 complexed with substrates that differ in size (32). Significant alterations in the conformation of the helix F/G region are also evident for complexes of CYP119 with imidazole or phenylimidazole (33). Substantial movement of the helix F/G region relative to the heme binding site is expected to occur in structures that exhibit an open conformation that would allow substrate entry and product egress as illustrated by the recent structure of rabbit CYP2B4, which displays an open cleft formed by helix B' and the helix F to helix G region (20). Additional structures of CYP2C8 complexed with various substrates will be required to reveal the extent of structural alterations that result from and contribute to substrate binding.

The flexibility of helix F/G region is likely to be reduced in CYP2C8 by formation of the dimer observed in this study, as illustrated by the relatively low temperature factors (Fig. 1A). The dimerization of CYP2C8 appears to be promoted by the presence of two molecules of palmitate that bind in the interface. Oligomerization states and the binding of molecules at external binding sites could both influence the shape of the substrate binding site through their effects on the conformation and flexibility of the helix F to helix G region. This raises the possibility that these factors could influence P450-catalyzed reactions and cooperative substrate-substrate interactions.

Both molecules of the CYP2C8 dimer form the fatty acid binding site. The hydrophobic interactions with the fatty acid are largely provided by a single P450 molecule and result from the hydrophobic surfaces of helices F', G', and G, which are arranged to form a tubular binding site for the hydrocarbon chain of the fatty acid. The binding of the carboxylate is stabilized by the dipole of helix G of the second CYP2C8 molecule in the dimer and direct hydrogen bonding interactions with the backbone amides in the first turn of helix G. The fatty acids appear to bind to CYP2C8 in the expression host. Structures of other proteins expressed in E. coli that have co-purified with palmitic acid bound have been described (34, 35). The CYP2C8 dimer persists through the purification of the enzyme suggesting that it is very stable.

A remarkable aspect of the geometry presented by the CYP2C8 dimer structure is that the N-terminal signal anchor domains are oriented on the same side of the complex (Fig. 1A). In addition, the proximal surfaces of both molecules remain exposed and available for interaction with P450 reductase. This suggests that the homodimer should be functional. Oligomerization and aggregation have been observed in solution for other microsomal P450s that have been expressed without their N-terminal signal sequences. This has generally been attributed to the hydrophobicity of the protein surface that is normally juxtaposed to the membrane surface and that contributes extensively to the binding of the truncated proteins to phospholipid membranes. The full-length proteins generally form larger aggregates that are likely to reflect interactions of the hydrophobic transmembrane domains. Several lines of evidence have suggested that P450s may form oligomers in their native, membrane-bound context. This possibility has largely been inferred by indirect methods such as the interpretation of rotational correlation times derived from spectroscopic studies (36, 37) and electron microscope immunolocalization (38). Cross-linking studies have also provided evidence for interactions between different P450s in the microsomal membrane (39). Very recently, fluorescent energy transfer experiments in intact cells have demonstrated that CYP2C2 fusion proteins with cyan and yellow fluorescent proteins form dimers in the endoplasmic reticulum of the cell in vivo (40). Interestingly, dimers were not observed for similar fusion proteins constructed with CYP2E1. The dimer in CYP2C2 may exhibit interactions between catalytic domains similar to those revealed by the CYP2C8 structure.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1PQ2 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health Grants GM31001 (to E. F. J.) and GM59229 (to C. D. S.). 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. 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. 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: P450 or CYP, generic terms for cytochrome P450 monooxygenases. Individual P450 enzymes are denoted by a number/letter/number combination derived from a uniform nomenclature based on sequence identity. Back


   ACKNOWLEDGMENTS
 
We thank the staff of the Stanford Synchrotron Radiation Facility for their generous assistance. Bill Webb and Gary Suizdak (Center for Mass Spectrometry at The Scripps Research Institute) are acknowledged for helpful recommendations for the mass spectrometry analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. K. Yano, M. R. Wester, G. A. Schoch, K. J. Griffin, C. D. Stout, and E. F. Johnson
The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-A Resolution
J. Biol. Chem., September 10, 2004; 279(37): 38091 - 38094.
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M. R. Wester, J. K. Yano, G. A. Schoch, C. Yang, K. J. Griffin, C. D. Stout, and E. F. Johnson
The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-A Resolution
J. Biol. Chem., August 20, 2004; 279(34): 35630 - 35637.
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E. E. Scott, M. A. White, Y. A. He, E. F. Johnson, C. D. Stout, and J. R. Halpert
Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-A Resolution: INSIGHT INTO THE RANGE OF P450 CONFORMATIONS AND THE COORDINATION OF REDOX PARTNER BINDING
J. Biol. Chem., June 25, 2004; 279(26): 27294 - 27301.
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