Crystal Structure of Human Cytochrome P450 2D6 withPrinomastat Bound*

Received for publication, September 26, 2011, and in revised form, February 2, 2012 Published, JBC Papers in Press, February 3, 2012, DOI 10.1074/jbc.M111.307918

An Wang‡, Uzen Savas‡, Mei-Hui Hsu‡, C. David Stout§1, and Eric F. Johnson‡2

From the Departments of ‡Molecular and Experimental Medicine and §Molecular Biology, The Scripps Research Institute,La Jolla, California 92037

Background: P450 2D6 contributes significantly to the metabolic clearance of many drugs.Results: Binding of prinomastat to P450 2D6 reveals a distinctive active site topology.Conclusion: P450 2D6 structural flexibility contributes to its catalytic versatility.Significance: This structure will aid efforts to minimize the impact of genetic variation and drug-drug interactions for newdrugs.

Human cytochrome P450 2D6 contributes to the metabolismof >15% of drugs used in clinical practice. This study deter-mined the structure of P450 2D6 complexed with a substrateand potent inhibitor, prinomastat, to 2.85 A resolution by x-raycrystallography. Prinomastat binding is well defined by electrondensity maps with its pyridyl nitrogen bound to the heme iron.The structure of ligand-bound P450 2D6 differs significantlyfrom the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition ofthe structures reveals significant differences for � sheet 1, heli-ces A, F, F�, G�, G, and H as well as the helix B-C loop. Thestructure of the ligand complex exhibits a closed active site cav-ity that conforms closely to the shape of prinomastat. The clo-sure of the open cavity seen for the 2F9Q structure reflects achange in the direction and pitch of helix F and introduction ofa turn at Gly-218, which is followed by a well defined helix F�

that was not observed in the 2F9Q structure. These differencesreflect considerable structural flexibility that is likely to contrib-ute to the catalytic versatility of P450 2D6, and this new struc-ture provides an alternative model for in silico studies of sub-strate interactions with P450 2D6.

Human cytochrome P4503 2D6 mediates the principal routeof metabolic clearance for �15% of the 200 most marketed

drugs that are primarily cleared by P450-mediated hepaticmetabolism (1). P450 2D6 oxidizes a variety of anti-psychotic,anti-depressant, and anti-arrhythmic drugs, which reflects thecapacity of the enzyme to oxidize moderately sized, basic sub-strates (2). Genetic polymorphisms contribute to a wide varia-tion of 2D6 expression and activity, leading to four phenotypesas follows: poor, intermediate, extensive, and ultra-fast drugmetabolizers (1, 3). Drugs such as thioridazine, debrisoquine,phenformin, and captopril, which exhibit comparatively nar-row therapeutic windows, can be problematic when used by2D6 poor metabolizers, which represent �10% of the Cauca-sian population (3–5).A structure for P450 2D6, PDB4 2F9Q, crystallized without a

substrate bound in the active site was reported previously (6).This study sought to determine the structure of the enzymewith a substrate or inhibitor bound in the active site. P450s canexhibit significant differences in the active site architecturebetween ligand-bound and ligand-free states (7–9), and thiswas observed for P450 2D6 in this study.Moreover, the success-ful crystallization and determination of structures for P450s1A2 (10) and 1B1 (11) required the stabilization of the enzymeswith a ligand during isolation and crystallization to maintainnormal substrate binding as judged by spectral properties of theenzyme complexes. As described here, this was also evident forP450 2D6, and the enzyme was purified and crystallized withprinomastat bound in the active site. The resulting structurediffers significantly from the ligand-free structure and providesan alternative model for understanding substrate and inhibitorbinding to P450 2D6 as well as demonstrating the pliant activesite architecture of the enzyme.

EXPERIMENTAL PROCEDURES

Expression of Modified P450 2D6 in Escherichia coli—Toreduce aggregation and increase solubility, the amino acidsequence of the N-terminal trans-membrane helix of P450 2D6was removed by replacing the first 33 residues with a shorteramino acid sequence, MAKKTSSKGKL. Additionally, a four-histidine expression tag was added to the C terminus to facili-tate purification using nickel-nitrilotriacetate-agarose affinity

* This work was supported, in whole or in part, by National Institutes of HealthGrant GM031001 (to E. F. J.). This work was also supported by Pfizer GlobalResearch and Development. Portions of this work were carried out at theStanford Synchrotron Radiation Lightsource, a national user facility oper-ated by Stanford University on behalf of the United States Department ofEnergy, Office of Basic Energy Sciences.

The atomic coordinates and structure factors (code 3QM4) have been depositedin the Protein Data Bank, Research Collaboratory for Structural Bioinformat-ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence may be addressed: Dept. of Molecular Biology,The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB8, La Jolla, CA92037. Tel.: 858-784-8738; Fax: 858-784-2857; E-mail: dave@scripps.edu.

2 To whom correspondence may be addressed: Dept. of Molecular and Exper-imental Medicine, The Scripps Research Institute, 10550 N. Torrey PinesRd., MEM-255, La Jolla, CA 92037. Tel.: 858-784-7918; Fax: 858-784-7978;E-mail: johnson@scripps.edu.

3 CYP or P450 is a generic term for a cytochrome P450 enzyme, and individualP450s are identified using a number-letter-number format based onamino acid sequence relatedness. 4 The abbreviation used is: PDB, Protein Data Bank.

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chromatography. These changes were introduced byamplification of the P450 2D6 cDNA with upper primer,5�-CGGAATTCCATATGGCTAAGAAAACGAGCTCTA-AACCACCAGGCCCCCTGCCAC-3�, and lower primer,5�-CGG AAT TCC GAA GCT TTC AGT GGT GGT GGTGGC GGG GCA CAG CAC AAA GCTC-3�. The PCRemployed 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 68 °Cfor 120 s using high fidelity Pfu Ultra (Agilent). In all otherrespects, the expression cassette corresponded to the codingsequence for the major human allele, CYP2D6*1. Theexpression cassette was inserted between the NdeI andHindIII restriction sites of the pCWori vector (12, 13). As anNdeI site was present in the coding sequence for P450 2D6,two fragments were generated from the PCR product byNdeI/BsmI and BsmI/HindIII digestion, which were com-bined with the NdeI/HindIII-digested vector backbone in asingle ligation reaction to produce the expression vector.The construct was verified by sequencing the expressioncassette.E. coli strain DH5� was transformed with both the CYP2D6

expression plasmid and the pGro7 plasmid for elevated expres-sion of the chaperone proteins GroEL and GroES (Takara BioInc., Shiga, Japan). The selected and validated transformantwasgrown in 500 ml of terrific broth containing ampicillin andchloramphenicol at 37 °C, 220 rpm in a tabletop C24KC refrig-erated incubator/shaker (New Brunswick Scientific, Edison,NJ) until an absorbance of �0.5 at a wavelength of 600 nm wasobtained. The temperature was lowered to 30 °C, and the incu-bation was continued at 190 rpm. After about 30min, when theabsorbance at 600 nm was �0.7–0.8, �-aminolevulinic acid (5mM), isopropyl �-D-thiogalactopyranoside (1 mM), and arabi-nose (4 g/liter) (Sigma) were added to induce the expression ofP450 2D6 and of the chaperones GroEL and GroES. Cells wereharvested after 24 h.Purification of P450 2D6—For protein extraction, sphero-

plasts were prepared as described (14) and suspended in a 500mM potassium phosphate buffer, pH 7.4, containing 20% glyc-erol, v/v, 0.2 mM prinomastat (Pfizer Global Research andDevelopment, La Jolla), 10 mM �-mercaptoethanol, 14 mM

CHAPS (Anatrace, Maumee, OH), and 1mM phenylmethylsul-fonyl fluoride. P450 2D6 was purified from the protein extractby nickel-nitriloacetate-agarose (Qiagen, Valencia, CA) affinitychromatography. After several washes, the protein was elutedusing a 10 mM potassium phosphate buffer, pH 7.4, containing30 mM histidine, 1 M NaCl, 0.05 mM prinomastat, 14 mM

CHAPS, 10 mM �-mercaptoethanol, 1 mM phenylmethylsulfo-nyl fluoride, and 20% v/v glycerol. The pooled fractions weredialyzed overnight against the same buffer with the NaCl con-centration lowered to 150 mM and without histidine beforeapplication to a column containing hydroxylapatite-agarosebeads (HA Ultrogel, BioSepra Inc) equilibrated with the samebuffer. The protein was eluted in 120mMpotassiumphosphate,pH 7.4, containing 20% v/v glycerol, 0.05 mM prinomastat, 10mM �-mercaptoethanol, 14 mM CHAPS, and 1 mM phenyl-methylsulfonyl fluoride. The protein solutionwas concentratedto 0.68 mM for crystallization using an Amicon ultracentrifugalfiltration device with a 50K molecular weight exclusion limit(Millipore). P450 concentrations were determined by CO-dif-

ference spectroscopy using an extinction coefficient of 0.091�M�1 cm�1 (15). As prinomastat reduces the formation of theCO complex, concentrations of the purified P450 2D6 prino-mastat complex used for crystallization were estimated by theintensity of the Soret absorption band. An extinction coeffi-cient of 0.113 � 0.006 �M�1 cm�1 was estimated for the com-plex by titration of the ligand-free enzyme with prinomastat asdescribed below. The extinction coefficient was calculated bydividing the absorbance of the complex observed at saturatingconcentrations of prinomastat by the concentration of theligand-free enzyme determined by CO-difference spectros-copy. The mean and standard deviation are reported for sevenreplicate experiments. Protein purity was assessed by SDS-PAGE followed by staining with Coomassie Brilliant Blue.Characterization of Ligand Binding by Visible Absorption

Spectroscopy—Binding constants were estimated by monitor-ing the concentration-dependent effects of ligands on the visi-ble absorption spectrum of the modified P450 2D6. For com-parison, full-length P450 2D6 was expressed in E. coli, andmembranes containing P450 2D6 were isolated (16). Stocksolutions of thioridazine hydrochloride and quinidine sulfatewere dissolved in water. Prinomastat was dissolved in DMSO,and the final concentration of DMSO in titrations was �1%.Difference spectra were calculated by subtracting the absorb-ance spectrum determined in the absence of the ligand fromspectra obtained with ligand added to the sample and referencecuvettes in equal amounts. Alternatively, difference spectrawere determined directly by addition of equal volumes of theligand to the sample cuvette and of the solvent to the referencecuvette, where both cuvettes contained the solution of the pro-tein. Peak to trough differences for the Soret absorption bandwere calculated and plotted against the concentration of theligand. Dissociation constants, Kd, and maximum absorbancechanges, �Amax, were estimated by nonlinear regression fittingof the results with a one-site ligand binding model. Kd and�Amax are reported as means � S.D. determined from at leastthree experiments. Alternatively, when the protein concentra-tion was near or exceeded the apparent Kd value, the quadraticform of the binding equation was employed with the assump-tion that the maximal change in absorbance reflects a 1:1 stoi-chiometry, andwhere P is the concentration of P450 2D6, and Lis the total concentration of the ligand as shown in Equation 1.

�A ��Amax�P � L � Kd � ��P � L � Kd

2 � 4PL

2P(Eq. 1)

Protein Crystallization—The modified P450 2D6 prinomas-tat complex was crystallized by hanging drop vapor diffusion.The drop contained 1 �l of the concentrated protein solution(620�MP450), 0.25�l of 70mMHEGA-10detergent (Anatrace,Maumee, OH), 0.625 �l of the protein buffer containing 0.01mM prinomastat without CHAPS, and 0.625 �l of precipitantsolution containing 20%w/v PEG-3350 and 0.1 M sodium caco-dylate, pH 7.0, and the drop was set to equilibrate at 296 Kagainst 0.5 ml of reservoir solution composed of the same pre-cipitant solution containing 6% v/v glycerol.Structure Determination—A dataset collected from a single

crystal of P450 2D6 complexed with prinomastat was used for

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structure determination. The crystal was flash-frozen in liquidnitrogen, and the data were collected at 100 K using the Stan-ford Synchrotron Radiation Lightsource beamline 7-1. MOS-FLM and SCALA (17) were used to index, integrate, and scalethe data. The structure was solved by molecular replacementusing PHASER (18). The model of the 2D6 prinomastat com-plex (PDB code 3QM4) was built using COOT (19) and refinedusing CNS (20) to 2.85 Å in the C2 space group with two mol-ecules in the asymmetric unit. Noncrystallographic symmetryrestraints were used initially. At later stages of refinement,these restraints were removed for divergent regions. The finalstructural model of P450 2D6 prinomastat complex encom-passes residues 33–497 of the protein for chain A. There wasinsufficient density tomodel theN-terminal residues precedingLeu-33 of chains A and B and residues 230–237 between heli-ces F� andG of chain B. Prinomastat wasmodeled in the activesite pocket above the heme prosthetic group in each chain. Anickel ion, identified by x-ray fluorescence, was bound on thesurface of chain B. Additionally, seven water molecules wereadded in the final stages of refinement. Data processing andmodel refinement statistics are listed in Table 1.

RESULTS

Protein Design—P450 2D6 was modified for expression inE. coli and to facilitate purification and crystallization. Thesechanges entailed removal of the N-terminal transmembraneleader sequence to improve solubility and to reduce aggrega-tion. This was accomplished by replacing the codons for aminoacid residues 2–33 of a cDNA encoding the major CYP2D6allele (UNP: P10635-1) with selected codons encoding a shorthydrophilic sequence AKKTSSKGKL as previously employedfor the crystallization of other mammalian microsomal P450s(21–24). Additionally, four codons for histidine were insertedupstream of the translation stop site as an expression tag foraffinity chromatography. The resulting construct is similar indesign to the one used by Rowland et al. (6) to determine thestructure of P450 2D6 in the absence of a bound ligand (PDBcode 2F9Q). The coding region of the latter construct differedby using a cDNA for a rare allelic variant of P450 2D6 (25, 26)exhibiting aV374M substitution and by the introduction of twoadditionalmutations, L230D and L231R, that increased the sol-ubility of the protein (6).Effects of Substrates and Inhibitors on the Ligand Binding

Properties of Modified P450 2D6—For our studies, initial pro-tein extracts were prepared by lysis of spheroplasts in bufferscontaining a substrate or inhibitor of the enzyme as well as thedetergent CHAPS. The detergent was necessary to maintainsolubility of the protein during purification. When prepared inthis manner, P450 2D6 exhibited substrate-dependent differ-ences in its absorption spectrum indicative of substrate or

FIGURE 1. Effects of ligands on the visible absorption spectra of P4502D6. A, spectra of P450 2D6 isolated in the presence of 50 �M thioridazine(blue), quinidine (green), or prinomastat (red) or in the absence of an exoge-nous ligand (black) are shown. For comparison, spectral absorbance isexpressed as millimolar absorptivity for each complex, and the four spectrawere normalized to each other at 477 nm to reduce the effects of experimen-tal variation. The scale for the apparent millimolar absorptivity is shown onthe left for wavelengths below 477 nm and on the right for longer wave-lengths. The thioridazine and quinidine complexes show an increase in thehigh spin state of the enzyme as judged by the increased absorbance at 390and 650 nm as well as decreases in the absorbance at 418 and 568 nm. Incontrast, prinomast produces a low spin enzyme as judged by the loss of the650 nm band and shift of the Soret band to 421 nm. B, concentration-depen-dent effects of thioridazine on the spectrum of the 10 �M P450 2D6 preparedin the absence of an exogenous ligand are displayed. Thioridazine in waterwas added to the sample and reference cuvettes in small increments to finalconcentrations of 10, 21, 31, 40, 50, 60, 70, 80, 99, 149, 198, and 247 �M. Thespectrum (blue) of P450 2D6 isolated in the presence of 50 �M thioridazineexhibits a larger proportion of high spin enzyme than evident for P450 2D6

prepared in the absence of an exogenous ligand at saturating concentrations.C, concentration-dependent effects of prinomastat on 10 �M P450 2D6 pre-pared without exogenous ligands are depicted. The final concentrations ofprinomast were 2, 4, 8, 16, 32, 64, and 127 �M. The maximum concentration ofDMSO was 0.66% v/v. The arrows indicate the direction of change in responseto addition of each ligand in B and C. Prinomastat converts P450 2D6 to a lowspin form similar to that isolated in the presence of prinomastat. Estimates ofthe Kd and �Amax for multiple determinations are reported in the text.

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inhibitor binding. As shown in Fig. 1A, when themodified P4502D6 was isolated in the presence of 50 �M quinidine (greenspectrum), the shape of the Soret band exhibits a shoulderindicative of increase in the proportion of the high spin staterelative to P450 2D6prepared in the absence of quinidine (blackspectrum). A larger increase in the proportion of high spin P450is evident when the enzyme is isolated in the presence of 50 �M

thioridazine (Fig. 1A, blue spectrum). The thioridazine complexexhibits a broad bifurcated peak indicative of roughly equalamounts of high and low spin heme iron. Ligand-dependentincreases in the proportion of high spin heme iron are generallytermed type 1 spectral changes and are thought to reflect per-turbations of water binding to the iron. In contrast, the prino-mastat complex displays a spectrum for a predominantly lowspin P450 indicative of the coordination of the prinomastatpyridyl nitrogen to the heme iron (Fig. 1A, red spectrum). Thisis generally described as a type 2 spectral change.Thioridazine is a 2D6 substrate that was reported to exhibit

Ki values of 0.75 �M (27) and 1.4 � 0.8 �M for competitiveinhibition of P450 2D6-catalyzed reactions (28). When thior-idazine was added to membranes isolated from E. coli-express-ing full-length P450 2D6, a type 1 spectral change was observedby difference spectroscopy with an apparent Kd of 1.4 � 0.53�M, which is concordant with the reported Ki values. In con-trast, when the enzyme isolated in the absence of an addedligand was titrated with thioridazine (Fig. 1B), the maximalchange in the absorbance was less than that observed for theenzyme isolated in the presence of thioridazine, and the appar-ent Kd of 39.6 � 1.8 �M was much higher than exhibited by thefull-length membrane-bound P450 2D6. The difference in theabsorption exhibited by thioridazine complex in the presenceof 50�M ligand and the ligand-free enzyme is 67.8mM�1 cm�1,which is greater than the estimated �Amax of 41.5 � 3.2 mM�1

cm�1 from the binding curve observed, which is not reached, inturn, until much higher concentrations of thioridazine areadded.Although the extent of the type 1 spectral change and the

binding affinity were diminished, the yields of the modifiedP450 2D6 in the absence of an exogenous ligand were similar tothose for the ligand complexes, and there was no evidence of asignificant conversion of the ligand-free preparation to an inac-tive form exhibiting a Soret absorption maximum 420 nm forthe ferrous enzyme. Although the apparent increase in Kd val-ues could reflect the binding of unknown competitor to theprotein, removal of ligands following isolation resulted in con-version of the modified P450 2D6 to a low affinity form.Similarly, titration with quinidine of modified P450 2D6 iso-

lated in the absence of an exogenous ligand exhibited a dimin-ished type 1 spectral change, �Amax of 8.5 � 3.0 mM�1 cm�1,when compared with the difference of 15.5 mM�1 cm�1

observed for the spectrum of the complex and the spectrum ofthe ligand-free enzyme (Fig. 1A). Moreover, the apparent Kd of42.1 � 1.4 �M is also much greater than reported for type 1spectral changes exhibited by the full-length P450 2D6expressed in E. coli by Hanna et al. (29), who estimated a Kd of0.7 �M, for the full-length enzyme expressed and purified fromE. coli and for the full-length enzyme expressed in yeast micro-somes, which exhibited an estimated Kd of 0.1 �M (26).

Hanna et al. (29) reported previously that quinidine did notproduce a type 1 spectral change with a similarly truncatedP450 2D6 construct. Although the type 1 spectral change wasnot observed by Hanna et al. (29), they found that the purified,truncated enzyme could be reconstituted with reductase andphospholipids and that the active reconstituted enzyme wasinhibited by quinidine at a concentration of 1 �M. Hanna et al.(29) speculated that the absence of the spectral change for thetruncated enzyme might reflect a more open conformation forthe truncated enzyme that might diminish the effects of thequinidine on the binding of water to the heme iron withoutindicating whether or not quinidine occupied the active site.Increased conformational dynamics might also contribute tothe reduced extent of the type 1 spectral change and to thediminished binding affinity observed in our studies. This expla-nation is consistent with recent molecular dynamics simula-tions for P450 2C9 indicating that the structure of N-terminallytruncated 2C9 exhibits a larger range of conformationaldynamics in solution and for the opening and closing of solventaccess channels during simulations than observed for a modelof the full-length P450 enzyme embedded in a phospholipidbilayer. Extensive interactions of the distal outer surface of thecatalytic domain with themembrane bilayer appear to limit theconformational dynamics of the protein in these simulations.Additionally, a bound substrate reduced the conformationaldynamics seen in the simulations (30, 31). Our observation thatligand complexes exhibited expected spectral changes whenisolated in the presence of the ligand suggested that isolation ofmodified P450 2D6 in this manner would be more suitable forcrystallization of the complexes. Additionally, it allowed us tomonitor the effects of different isolation protocols and condi-tions on the stability of the complex.Colleagues at Pfizer, Inc. (La Jolla, CA) suggested that prino-

mastat, an experimental matrix metalloprotease inhibitor,might provide a good stabilizing ligand for our studies and pro-vided us with sufficient quantities of prinomastat for use inpurification and crystallization of P450 2D6. The Pfizer scien-tists had observed that prinomastat is a potent inhibitor of P4502D6 with an observed Ki � 0.049 �M and that prinomastatproduced a type 2 binding spectrumwith an apparentKd of 0.29�M with purified E. coli-expressed full-length P450 2D6obtained from the commercial supplier Panvera.5 Our studieswith membrane-bound E. coli-expressed full-length P450 2D6confirmed the type 2 change with an estimated Kd �0.02 �M,the upper 95% confidence value. The binding of prinomastatshifts the Soret maximum to a longer wavelength (422 nm) anddecreases the intensities of the absorbance bands at 568 and 650nm (Fig. 1, A (red) and C). In this case, the spectrum of theenzyme obtained following titration of the modified P450 2D6(Fig. 1C) is highly similar to the spectrum obtained for theenzyme isolated in the presence of 50 �M prinomastat (Fig. 1A(red spectrum)), indicating that pyridyl nitrogen binds to theheme iron in both cases. Nevertheless, the estimated Kd of1.96� 1.06 �M is higher than theKd value determined spectro-scopically for the full-length enzyme.

5 Michael Zientek (Pfizer, Inc., La Jolla, CA), personal communication.

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In contrast, the affinity of the isolated thioridazine complexis higher for prinomastat than for the enzyme purified in theabsence of a ligand even though thioridazine is present to com-pete for binding. As shown in Fig. 2A, 100-fold dilution ofhighly concentrated solution of the thioridazine complex in thi-oridazine-free buffer to a final concentration 4.6 �M does notsignificantly reduce the high spin component. At this dilution,the concentration of thioridazine is predicted to be a 5.1�M-based saturation of a single binding site in the complex anda 100-fold dilution of the 50 �M thioridazine in the buffer. Anincreased absorbance seen at 310 nm is consistent with thisestimate for thioridazine. Under this condition, titration of theprotein with prinomastat converts the spectrum to that of theprinomastat complex (Fig. 2A). A fit of the quadratic form ofthe one-site binding equation by nonlinear least squares regres-sion (Fig. 2C), yields an apparent Kd of 0.62 � 0.06 �M. As thepresence of residual thioridazine competes for binding, the actualKd value for prinomastatwill be lower. TheKdwas estimated to be0.19 � 0.02 �M using DynaFit (32, 33) when competitive bindingby 5.1 �M thioridazine was included in the model with a Kd of 1.4�M, which is the Kd value observed for the full-length membraneenzyme. When the concentrated thioridazine complex is diluted1000-fold to a final concentration of 0.46 �M, the predicted con-

centration of thioridazine is diminished to 0.51 �M. At this dilu-tion, a reduction in theproportionofhighspinP450 is evident (Fig.2B), indicative of dissociation of some thioridazine, which is con-sistent with the Kd value of 1.4 � 0.53 �M observed for the full-length enzyme. In this condition, titration with prinomastat con-verts themodified P450 2D6 to the low spin prinomastat complex(Fig. 2B), with an apparent Kd of 0.064 � 0.025 �M determinedfrom the fit of the binding equation to these results (Fig. 2D).Withreduced residual thioridazine, this value approaches more closelyto the Kd blue exhibited by membrane-bound full-length P4502D6.Based on these observations,modified P450 2D6was isolated

in the presence of ligands for crystallization to maintain thenative ligand binding properties of the enzyme. As the P4502D6 prinomastat complex crystallized more readily than othercomplexes, conditions were optimized to produce crystals thatdiffracted to better than 2.85 Å. The structure of the prinomas-tat complex was determined for a dataset collected from a sin-gle crystal exhibiting the C2 space group with twomolecules ofthe complex comprising the asymmetric unit. Model refine-ment statistics are shown in Table 1. The resulting structuredefines the binding interactions of prinomastat with theenzyme, and when compared with the 1F9Q structure of P450

FIGURE 2. Concentration-dependent effects of prinomastat on P450 2D6 isolated in the presence of 50 �M thioridazine. The concentrated P450 2D6thioridazine complex in buffer containing 120 mM potassium phosphate, pH 7.4, 20% v/v glycerol, 10 mM �-mercaptoethanol, 14 mM CHAPS, and 50 �M

thioridazine was diluted into buffer containing 120 mM potassium phosphate, pH 7.4, and 20% v/v glycerol. A, prinomastat in DMSO was added in smallincrements to final concentrations of 1, 2, 4, 8, 16, 32, and 64 �M to 4.6 �M P450 2D6 thioridazine complex (100-fold dilution). The maximum concentration ofDMSO was 0.07% v/v. The scale for the apparent millimolar absorptivity is shown on the left for wavelengths below 477 nm and on the right for longerwavelengths. B, prinomastat was added to 0.46 �M of the P450 2D6 thioridazine complex (1000-dilution). Only the Soret region is shown. The proportion ofhigh spin P450 is reduced at this concentration of the protein based on the lower ratio of absorbance at 390 nm relative to 424 nm. The final concentrations ofprinomastat were 0.1, 0.2, 0.4, 0.8, 1.8, 3.8, and 7.8 �M. The maximum concentration of DMSO was 0.02% v/v. The arrows indicate the direction of change inresponse to addition of each ligand in A and B. Difference spectra were calculated for the spectra in A and B, and the differences of absorbance between 424and 390 nm were plotted versus the final concentration of prinomastat in C and D, respectively. The binding curves were computed by nonlinear least squaresregression to estimate values of �Amax and Kd using the quadratic form of the binding equation. Estimates of the Kd values for multiple determinations arereported in the text.

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2D6 isolated and crystallized in the absence of ligand, the pri-nomastat complex exhibits a distinctly different conformationfor the active site cavity and for the tertiary and secondarystructure forming the distal portion of the active site.Prinomastat-P450 2D6 Interactions—The binding of prino-

mastat in the active site of P450 2D6 was well defined by a2�Fo� � �Fc� �A-weighted electron density map calculated for amodel that did not include prinomastat (Fig. 3A). The pyridylnitrogen is located 2.2Å from the heme iron consistentwith theobservation that prinomastat shifts the Soret maximum to 422nm as typically seen for nitrogenous ligands that coordinate tothe heme iron. Although prinomastat has two H-bond donorsites and six potential H-bond acceptor sites, excluding the pyr-idine, only three hydrogen bonding interactions are evidentbetween P450 2D6 and prinomastat. One hydrogen bond isevident between Gln-244 and one of the prinomastat sulfonyloxygens. Another hydrogen bond is located between the amidehydrogen of the hydroxamic acid moiety and Ser-304, and thethird hydrogen bond occurs between the hydroxyl group of thehydroxamic acid moiety and Asp-301. Although many P4502D6 substrates contain basic nitrogens that are positivelycharged at neutral pH, as recently reviewed (2), prinomastat isneutral as neither the sulfonamide or the pyridine are likely tobe protonated, and the hydroxamic acid moiety is not ionized.With the exception of Glu-216, the remaining contactsbetween the protein and prinomastat are nonpolar (Fig. 3A).Structural Similarities and Differences between the 3QM4

and 2F9Q Structures of P450 2D6—The structure of the P4502D6 prinomastat complex defines portions of the structure thatwere not modeled in the 2F9Q structure, such as the loopbetween the proline-rich motif and helix A. Additionally, resi-dues 229–239 of the 2F9Qmodel were modeled as alanine res-idues because side chains were not defined by electron densitymaps (6). This region could be modeled completely for chain A

FIGURE 3. A, amino acid side chains (cyan carbons) contacting prinomastat (green carbons) are depicted along with the heme prosthetic group (pink carbons) asstick figures. The gold mesh depicts a 2�Fo� � �Fc� �A-weighted prinomastat omit map contoured at 1� around the prinomastat molecule. Dashed lines identifyhydrogen bonds and the coordination of the pyridyl moiety with the heme iron (orange sphere) with the interatomic distances shown. Amino acid residues areidentified by single letter amino acid codes and residue number. Nitrogen, oxygen, sulfur and iron atoms are colored blue, red, yellow, and orange, respectively.B, ribbon diagram showing the superposition of the structure of the prinomastat complex 3QM4 with the substrate-free structure 2F9Q (carbons colored lightmagenta). Helices are designated by letters and sheets by numbers. The arrow indicates helix F� seen in the structure of the prinomastat complex. Thesurrounding region exhibits significant structure differences. C, comparison of the shapes of the active site cavities is depicted by a mesh surface colored greenfor the prinomastat complex and light magenta for 2F9Q structure. Cavity surfaces were generated by VOIDOO using a 1.4-Å probe. Water molecules were usedto terminate the exterior opening of the solvent channel seen for the 2F9Q structure (arrow).

TABLE 1Data collection and refinement statistics

1 Values for the highest resolution shell, 3.00 to 2.85 Å, are shown in parentheses.2 Ramachandran plot indicates 89.2% of residues in most favored regions, 98.7% inallowed regions, and 1.3% in disfavored regions.

3 The full sequence corresponds to residues 23–497 when numbered according tothe native protein. There was insufficient density to model residues 23–32 forchains A and B and residues 230–237 for chain B.

4 A nickel ion is coordinated with His-258(B) on the protein surface in the inter-face with a symmetry related copy of chain B.

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of the prinomastat complex, but it could not be modeled forresidues 230–237 of chain B.The most prominent difference between the two structures

is the presence of a distinct F� helix, residues 218–225, in thestructure of the prinomastat complex that is almost perpendic-ular to the axis of helix F, residues 199–215 (see arrow in Fig.3B). In contrast, helix F�was not observed in the 2F9Q structureof P450 2D6, where a continuous helix encompasses residues199–225. This long helix passes over helix I in a direction thatpasses under the location of helix F� of the prinomastat com-plex. As a result of this difference seen for the position of helixF�, there are substantial changes evident for adjacent structuralcomponents that include helix A, the loop between the first twostrands of sheet�1 (residues 72–77), and helices F, G, G, andH(Fig. 3B).Helix F� is a common feature in structures of family 2 P450s

with a turn between helix F and helix F�. Ser-217 and Gly-218contribute to the flexibility of the polypeptide backbone in thisturn because of the small size of the side chain and absence ofside chain, respectively. The corresponding amino acids thatform this turn are similar for other family 2 P450s. The positionof helix F� in the prinomastat complex is similar to that seen forthe structures 2A6 (PDB code 1Z10) and 2C8 (PDB code 2NNJ)as are the positions of helices F andG, the first turn in�-sheet 1,and the N-terminal region before helix A.An unusual feature of the 2F9Q structure noted by Rowland et

al. (6) is a relatively shortGhelix comparedwithotherP450 family2 structures. The shortened helix G is preceded by another shorthelix, which these authors designated G�. As this region is nor-mally part of a longer G helix in other family 2 P450s, we havedesignated it as helix G in Fig. 3B. This unusual feature is con-served for both the 2F9Q and 3QM4 structures. In the prinomas-tat complex, an almost anti-parallel orientation of the helix F� andhelix G is seen. The loop between the two helices corresponds tothe portion of the polypeptide chain that forms helix G� in struc-tures of other family 2 enzymes, such as P450s 2A6 and 2C8. Thisloop and its side chains are defined in chainAbut not in chainBofthe 3QM4 structure. This loop does not display a well definedhelical structure. A structure-based sequence alignment indicatesthat the loopappears tohaveone lessaminoacid residuerelative tothe other family 2 P450s.Active Site Architectural Differences—These differences of

tertiary structure are associated with distinctly different shapesfor the active site cavities of the prinomastat complex and the2F9Q structure as depicted by solvent-accessiblemesh surfacesshown in Fig. 3C. In the prinomastat complex, helix F and sheet�-4 are positioned to close an open solvent channel evident for2F9Q structure. The channel passes under helix F and betweenhelix I and sheet �-4. Additionally, the orientations of helices Fand G increase the height of the active site cavity above theheme to better accommodate prinomastat with its pyridylnitrogen oriented for coordination to the heme iron. Thecumulative effects of these changes increase the volume of theactive site cavity relative to that of the 2F9Q structure, 712versus 582Å3 as calculated using VOIDOO (34). As a result, thecavity of the 3QM4 structure is distributed more uniformlyabove the surface of the heme.

The different cavity shapes and volumes also reflect differ-ences in the position and orientation of amino acid side chainslining the cavities in the two structures. This is illustrated forthe sector of the cavity formed by the intersection of the B-Cloop andhelicesG and I in Fig. 4A. The differences are relativelysmall for residues on helix I with the exception of a distortion inthe helix that accommodates the prinomastat pyridyl groupbound to the heme iron. This distortion leads to significantchanges in the positions of Ser-304, Ala-305, Val-308, and Thr-309 with largest differences, �1.9 Å, seen for Ala-305 and Thr-309, which contact the pyridyl group of prinomastat. In con-trast, Ala-300 and Asp-301 on helix I and Phe-120 on theadjacent helix B-C loop are not greatly affected. Nevertheless,larger differences of �1.5–1.9 Å are seen for Leu-110 and Phe-112 on the helix B-C loop, whichmoves in to contact prinomas-tat. The largest differences are evident for residues on helix G,which exhibit �2-Å differences for C� positions of Gln-244,Phe-247, and Leu-248, and in the case of Gln-244, there is alsoa significant change in the side chain rotamer to accommodatehydrogen bonding to the sulfonyl oxygen of prinomastat.Similarly, when the sector of the cavity formed by the intersec-

tion of �-sheet 4 with helices F and I is examined (Fig. 4B), thelargest changes in amino acid side-chain positions occur in theupper portion of the cavitywith smaller changes observed forVal-370 near the base of the cavity and the heme surface. ResiduesAla-209, Leu-213, andGlu-216onhelix F are shifted to expand thecavity to accommodate prinomastat with the position of the car-boxylate moiety of Glu-216 displaced by more than 4 Å. In con-trast, the side chain of Phe-483 on the turn in the adjacent sheet�-4 swings into the cavity by 4 Å to form favorable nonbondedinteractionswith prinomastat. The convergence of the amino acidside chains on helix F and sheet�-4 with those on helix I close theopen solvent channel evident in the 2F9Q structure.The largest differences for side-chain positions in the active

site reflect the different locations of the helix F� segment of thepolypeptide chain in the two structures (Fig. 4C). A turn follow-ing Glu-216 in the prinomastat complex terminates helix F andorients the helix F� region so that its helical axis points towardthe N-terminal side of the helix B-C loop (Fig. 3B). The polarsurface of the helix F� region, which is on the exterior surface ofthe 2F9Q structure (Fig. 4C), is rotated so that Glu-222 andArg-221 are directed into the interior of the protein in the pri-nomastat complex, where a substrate access channel passesbelow helix F� and above the surface of the heme and the loopbetween helix K and �-sheet 1. The outer surface of theentrance channel is formed by �-sheet 1, helix A, and the loopbetween the proline-rich region at the N terminus of themodeland helix A (Fig. 4D). This substrate access channel is not evi-dent for the 2F9Q structure where the extended and combinedhelices F and F� pass through this region. Glu-222 is likely toremain hydrated in the substrate access channel of the 3QM4structure. The access channel is closed off from the active siteby the close proximity of Glu-216 on helix F with the guani-dinium group of Arg-221 on helix F�, which face into the activesite cavity (Fig. 4C). These amino acid side chains together withthose of Val-104, Phe-120, and Leu-121 on the helix B-C loopand Phe-483 on the turn in sheet �-4 constrict the connectionbetween the entrance channel and the prinomastat binding

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cavity, creating a separate solvent-accessible antechamber (Fig.4, C andD). Additionally, the interaction between Arg-221 andGlu-216 provides a favorable electrostatic interaction betweenthe oppositely charged side chains in the interior of the protein,but the overall electrostatic potential remains rather negativebecause of the presence of Asp-301 and Glu-222. The constric-tion of the channel better accommodates the size and shape ofprinomastat and increases van derWaal’s contacts betweenpri-nomastat and the enzyme. Nevertheless, the overall flexibility,implied by comparison of the 3QM4 and 2F9Q structures, sug-gests that the constriction can open to form a passage to theoutside for substrates and products. As shown by the compar-isons in Fig. 4, these differences in the tertiary and secondarystructure of the prinomastat complex lead to significant shiftsin the positions of the contact residues relative to the positionsseen in the 2F9Q structure.

DISCUSSION

This study describes the first structure deposited in the PDBof humanP450 2D6 complexedwith a ligand. The structurewasdetermined with prinomastat bound in the active site. Prino-mastat is a matrix metalloprotease inhibitor that is primarilymetabolized to a pyridine N-oxide metabolite, and this meta-bolic pathway is thought to be mediated by P450 2D6 (35). Thex-ray diffraction data indicate that prinomastat binds to ferricP450 2D6 with the pyridyl moiety bound to the heme iron con-sistent with the observation of a red shift in the wavelengthmaximum of the Soret absorption band observed for the com-plex. In many cases, coordination of nitrogenous groups onsubstrates and inhibitors with the heme iron substantially con-

tributes to the stability of the complex when compared withclosely related analogs that do not ligate to the heme iron.Although this has often been considered a dead end inhibitorcomplex, recent studies have emphasized that these hemeligands are oxygenated at significant rates (36). Dissociation ofthe pyridyl group from the iron followed by oxygenation of thepyridyl group by the reactive intermediate formed by theenzyme would be consistent with the observed formation ofthe pyridyl N-oxide metabolite.

Our objective in determining the structure of the prinomastatcomplex was to determine likely differences relative to the struc-ture of the ligand-free protein that occur when prinomastat bindsin the active site of P450 2D6. Rowlands et al. (6) described theactive site cavity of the ligand-free 2F9Q structure as having theshape of a right foot with the heel positioned above the heme,the arch passing over Phe-120 toward Asp-301, and the calf pass-ingoutof the cavityunderhelixF (Fig. 3C). Extending this analogy,the shape could also be described as a right boot, as the cavity isopen and forms a cavity for substrate binding. These authors alsodiscussed docking of the substrate debrisoquine, 3,4-dihydro-1H-isoquinoline-2-carboximidamide (Mr � 175), in the active siteand indicated that some degree of protein rearrangement wasrequired for binding, as illustrated by a model obtained from amolecular dynamics simulation. This in silico model alloweddebrisoquine to be positionedwith its protonated 2-carboximi-damide moiety forming a favorable charge-charge interactionwith Asp-301 and the 3,4-dihydro-1H-isoquinoline ring posi-tioned for oxygenation by the heme-bound reactive intermedi-ate. This binding orientation was favored by changes in the

FIGURE 4. A and B show differences for structural features between the two proteins on the right and left sides, respectively, viewed across the heme towardhelix I. C shows the view looking across the heme to the side of the cavity opposite helix I with solvent-accessible surface of the antechamber for substratebinding depicted as a black mesh. D shows the ribbon diagram indicating the juxtaposition of the active site cavity above the heme and antechamber forsubstrate binding. The arrow indicates an open passageway from the surface to the antechamber. The coloring scheme is described in the legend to Fig. 2.

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position of Phe-120 and Phe-483 to open the heel and archareas of the cavity. Based on the 2F9Q structure and dockingstudies of Rowland et al. (6), it seemed likely that additionalchanges would be required to accommodate the larger prino-mastat molecule,Mr � 426. As shown in Fig. 3C, a shift in theposition of helices F and G is required to accommodate the sizeof prinomastat as well as coordination of the pyridyl moiety tothe heme iron, which requires the plane of pyridyl ring to adoptan almost perpendicular orientation relative to the plane of thehemewith the pyridyl nitrogen positioned above the heme iron.This closes the solvent channel formed by the top of the bootand expands the heel and arch regions of the cavity. The result-ing structure represents a tertiary structure of the enzyme thatcan accommodate large substrates and inhibitors in a closedcavity above the heme. Thus, the structure of the prinomastatcomplex, 3QM4, provides an alternative model for computa-tional examination of ligand interactions with P450 2D6 thatcomplements the existing 2F9Q structural model.It isunlikely thatoneormoreof the threeaminoaciddifferences

between the modified proteins used to determine the 3QM4 and2F9Q structures contributes directly to the observed differencesbetween the structures. TheV374Mmutation in the allelic variantused for the 2F9Q structure lies outside the active site cavity at thebase of the substrate access channel observed in the structure fortheprinomastat complex.Although thismutationhasbeen shownto alter catalytic properties for some substrates (25, 26), the largersize of the methionine side chain (Fig. 4C) appears to be easilyaccommodated in the substrate access channel observed in thestructure of the P450 2D6 prinomastat complex. The two addi-tional mutations, L230D and L231R, in the construct used fordetermination of the 2F9Q structure reside in a loop between hel-ices F� and G that typically correspond to helix G�. Althougheffects of these differences on protein folding and stability cannotbe easily inferred from these considerations, it would appear thatthese mutations could be accommodated easily in the 3QM4structure.The helix F�–G� region normally exhibits a reverse

amphipathicity that contributes to the formation of a hydro-phobic exterior surface that is thought to interact with themembrane bilayer, as reviewed in Ref. 30. In this respect, it isimportant to note that the polar surface of helix F� of P450 2D6is populated by the charged residues Glu-216, Arg-221, andGlu-222 that correspond to polar neutral residues for struc-tures of other family 2 P450s. The charged nature of these sidechains could contribute to the stability of the solvent-exposedorientation of this surface in the 2F9Q structure. This surface isoriented toward the interior of the protein in the 3QM4 struc-ture of the prinomastat complex, where Arg-221 forms a saltbridge with Glu-216 that stabilizes the buried location of thetwo residues in the prinomastat complex. Moreover, Glu-222resides at the entrance to the putative substrate access channel,where it remains hydrated in the 3QM4 structure.In contrast to prinomastat, many substrates and inhibitors of

P450 2D6 are thought to bind as positively charged species atneutral pH, and it has been proposed that the binding of thesepositively charged compounds is stabilized by the formation ofsalt bridges with Glu-216, Asp-301, or possibly Glu-222.Mutagenesis of these residues indicates that the acidic nature of

these side chains is important for maintaining efficient metab-olism of cationic substrates, as recently reviewed (2). The pres-ence of Asp-301 is not unique to P450 2D6 as a correspondingaspartate residue is found at this location in structures of 1A1,1B1, 2C5, 2C8, 2C9, and 2E1, which do not contribute substan-tially to the metabolism of cationic substrates. Charged sub-strates could encounter a kinetic barrier for binding that mightotherwise be stabilized by interactions with the conservedaspartic acid residue. Conversely, acidic residues correspond-ing to Glu-221 and Glu-216 are not found in these enzymes.Glu-216 is positioned, albeit differently, to support substratebinding in both the 3QM4 and the 2F9Q structures. Glu-222 islocated on the outside of the protein in the 2F9Q structure andin a putative substrate-binding entrance channel seen in the3QM4 structure. However, the presence of Glu-222 in a sub-strate access channel could play a role in substrate binding byproviding “bait” for the initial binding of positively charged sub-strates. This is consistent with the observation that the E222Amutant exhibits a greatly reduced Vmax without significantlyaffecting the Km value or enantiomer selectivity exhibited forbufuralol 1�-hydroxylation (37). In conjunction with Glu-216and Asp-301, Glu-222 likely facilitates substrate entry andproper orientation for metabolism followed by subsequenttransit further into the cavity for interaction with Glu-216and/or Asp-301 during catalysis.In summary, the 3QM4 structure is the first deposited in the

PDB for P450 2D6 complexed with a substrate or inhibitor. Asjudged by a comparison to ligand-free structure 2F9Q deter-mined by Rowland et al. (6), the binding of prinomastat in theactive site is associated with a number of changes in the struc-ture of the enzyme that were anticipated based on the size andshape of the compound and its coordination to the heme iron.This study reiterates the potential usefulness of employing vis-ible spectroscopy to monitor substrate and inhibitor bindingcontinuously during the purification of the enzyme and theselection of detergents for purification and crystallization. Thesubstantial differences exhibited by the 3QM4 and 2F9Q struc-tures provide two complementary models for computationalapproaches to examine substrate and inhibitor binding to theenzyme. Efforts are under way to obtain structures for addi-tional complexes to better define the contributions of the acidicresidues to P450 2D6 substrate binding interactions and todelineate adaptive changes in the active site for the binding ofstructurally distinct substrates and inhibitors.

Acknowledgments—The technical support of LacThu Tonnu for thepreparation and crystallization of P450 2D6 and the help provided bythe support staff at Stanford Synchrotron Radiation Lightsource fordata collection are greatly appreciated. We also thank Pfizer GlobalResearch and Development for providing prinomastat for the purifi-cation and crystallization of the prinomastat P450 2D6 complex andBen Burke, Caroline Lee, Michael Wester, and Michael Zientek forhelpful discussions during the course of this work. The Stanford Syn-chrotron Radiation Lightsource, Structural Molecular Biology Pro-gram, is supported by the United States Department of Energy, Officeof Biological and Environmental Research, and byNational Institutesof Health, NCRR, Biomedical Technology Program, and NIGMS.

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Human CYP2D6 Structure

MARCH 30, 2012 • VOLUME 287 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10843

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An Wang, Uzen Savas, Mei-Hui Hsu, C. David Stout and Eric F. JohnsonCrystal Structure of Human Cytochrome P450 2D6 with Prinomastat Bound

doi: 10.1074/jbc.M111.307918 originally published online February 3, 20122012, 287:10834-10843.J. Biol. Chem. 

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