THEJOURN/\l_ OF BlOl_OC1lCAl_ CHENUSTRY VOL. 28l, NO ll, pp 76l4 7622, March l7, 2006
`© 2006 by The Ariierrcarr Society for Biochemistry and Molecular Biology, lrir.
`Printed lil the U S A
`
`Crystal Structure of Human Cytochrome P450 2D6*‘
`Received for publicationoctober 16, 2005, and in revised form, November 22, 2005 Published,1BC Papers in Press, December 13, 2005, DO 10.1074/jbc.i\/lSl 1232200
`
`Paul Rowland”, Frank E. Blaneyl, Martin G. Smyth, Jo J. Jones, Vaughan R. Leydon, Amanda K. Oxbrow,
`Ceri J. Lewis, Mike G. Tennantg, Sandeep Modi, Drake S. Eggleston, Richard J. Chenery, and Angela M. Bridges‘
`From the Department of Discovery Research, Glaxo$mithKline, New Frontiers Science Park, Third Avenue,
`Harlow, Essex, CM 79 5A W, United Kingdom
`
`Cytochrome P450 2D6 is a heme—containing enzyme that is
`responsible for the metabolism of at least 20% of known drugs. Sub-
`strates of 2D6 typically contain a basic nitrogen and a planar arc-
`matic ring. The crystal structure of human 2D6 has been solved and
`refined to 3.0 A resolution. The structure shows the characteristic
`P450 fold as seen in other members of the family, with the lengths
`and orientations of the individual secondary structural elements
`being very similar to those seen in 2C9. There are, however, several
`important differences, the most notable involving the F helix, the
`PG loop, the B’ helix, ,8 sheet 4, and part of [3 sheet 1, all of which
`are situated on the distal face of the protein. The 2D6 structure has
`a well defined active site cavity above the heme group, containing
`many important residues that have been implicated in substrate
`recognition and binding, including Asp~301, Glu—216, Phe~48 3, and
`Phe-120. The crystal structure helps to explain how Asp~301, Glu—
`216, and Phe—483 can act as substrate binding residues and suggests
`that the role of Phe—120 is to control the orientation of the aromatic
`
`ring found in most substrates with respect to the heme. The struc—
`ture has been compared with published homology models and has
`been used to explain much of the reported site—directed mutagene-
`sis data and help understand the metabolism of several compounds.
`
`The cytochromes P4504 constitute a superfamily of heme—containing
`enzymes that catalyze the metabolism of a wide Variety of endogenous
`and xenobiotic compounds. This is accomplished through the activa—
`tion of molecular oxygen by the heme group, a process that involves the
`delivery of two electrons to the P450 system followed by cleavage of the
`dioxygen bond, yielding water and an activated iron—oxygen species
`(Compound 1), which reacts with substrates through a variety of mech—
`anisms (1).1n eukaryotic species, the electron source is a single flaVopro—
`tein, the FAD/FMN—containing cytochrome P450 reductase, which
`binds to the largely basic proximal face of the cytochrome through a
`number of salt bridges. Of the known human isoforms, cytochrome
`P450 ZD6 is responsible for the metabolism of at least 20% of known
`drugs (2), with only 3A4 being responsible for a higher
`(50%)
`percentage.
`
`5
`
`* 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 andstructure factors (code 2F9Q) have been deposited in the Protein
`Data Bank, Research Col/aboratory forStructural Bioinformatics, Rutgers University, New
`Brunswick, NJ {http://www.rcsb.org/).
`The on-line version of this article (available at http://www.jbc.org) contains supple-
`mental Figs. 14%.
`‘ These authors contributed equally to this work.
`2To whom correspondence should be addressed. Te|.: 44-1279-622997; Fax: 44-1279-
`627666; E-mail: Paul_2_Row|and@gsk.com.
`3 Present address: Pharmix Corp., 2000 Sierra Point Pkwy., Suite 500, Brisbane, San Fran-
`cisco, CA 94005.
`4:‘
`‘E
`.
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`P450 enzymes denoted by a number/letter/number combination derived from the
`sequence identity; SRS, substrate recognition site; SDM, site—directed mutagenesis.
`
`The CDNA encoding human P450 ZD6 has been characterized (3) and
`subsequently localized to chromosome 22 in the q13.1 region
`A
`relatively large number of genetic polymorphisms have been described
`for ZD6, some of which can either result in rapid or very poor metabo—
`lism. One well characterized allelic variant is responsible for a condition
`known as debrisoquine/sparteine type polymorphism (5, 6). This arises
`as a result ofvarious genetic mutations and affects a significant percent—
`age of the Caucasian population
`it results in the defective metabo~
`lism of a number ofimportant drug molecules, including debrisoquine,
`from which the condition got its name. The inability of patients to turn
`over compounds such as debrisoquine eventually leads to toxic levels of
`the drug in the body. Binding of any drug to these allelic 2D6 variants
`can cause drug—drug interactions, which can lead to severe side effects
`and has resulted in early termination ofseveral candidate drugs in devel—
`opment, refusal of regulatory approval, severe prescribing restrictions,
`and withdrawal of drugs from the market.
`\X/hereas 3A4 exhibits a wide diversity in its substrate recognition, a
`fact that is often attributed to its large cavity size (8), 2D6 generally only
`recognizes substrates containing a (protonated) basic nitrogen and a
`planar aromatic ring. These features are found especially in a large num—
`U‘
`
`
`
`[-A1is19Aiuf11[iq1aput2A112/31o'oqt‘mmm//zdnqmoi;papeoiumoq
`
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`grog‘g1aqu1aAoNuosautziqnBuuaaurfiug/aouaiog79[ea
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`protein~coupled receptor superfamily of proteins. For this reason, 2D6
`is the most widely studied isoform, both from experimental site~di—
`rected mutagenesis (SDM) studies and from computational modeling.
`With regard to the latter, numerous pharmacophore models have been
`described (9-11), but it has become clear that no single model can
`account for the diversity observed in the regioselectivity of substrate
`metabolism. Likewise, various homology models have been constructed
`(10 —24), but the sequence identity between ZD6 and the available x—ray
` r r the “(Q i ndody
`18% for 3A4. Thus, the availability of a crystal structure of ZD6 was
`anticipated to go a long way toward explaining the effects of polymor~
`phism and the results of SDM studies and toward answering some ofthe
`questions raised by in silico modeling work. Such improved information
`would in time help achieve the ultimate goals of predicting the meta»
`bolic fate of drug compounds or predicting which compounds would
`inhibit the cytochromes and eventually lead to improved therapeutic
`ligand design.
`Recently, the structures
`have been solved by x—ray crystallography. The first ofthese, rabbit 2C5
`(25), showed considerably higher homology to the human isoforms than
`had been found with previous bacterial enzyme structures and led
`quickly to a new generation of computational models (19 ~24). This has
`been followed by the reports of the crystal structures of the human 2C9
`(26, 27), 2C8 (28), 3A4 (29, 30), and 2A6 (31) isoforms. This was made
`possible by truncation of the membrane—bound N—terminal domain
`and, in some cases, the introduction of some small mutations that
`helped to solubilize the protein. in recent years, we have embarked on a
`similar approach, and in this paper we report the x—ray crystal structure
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`ofthe human cytochrome P450 2D6 at 3.0 A resolution. The solubilizing
`mutations were designed using an early model that was based on homol—
`ogy with cytochrome P450 BM3 (32). This new structure has already
`proved to be valuable in understanding the metabolism of several com»
`
`MATERIALS AND METHODS
`
`Cytochrome P450 2D6 Crystal Structure
`
`inoculation, the culture was grown at 30 “C with an airflow rate of 1.5
`liters air/min and an agitation speed of 680 rpm until induction. Induc—
`tion was at A600 0.8 with 0.5 mM isopropyl 1—thio—[3—D—galactopyrano—
`side and 0.5 mM 5~aminolevulinic acid. Following further incubation at
`the samec cellswere
`P450 concentration was determined by CO difference spectrum using
`whole cells.
`
`Cell L)/si’s——Frozen pellets were resuspended in lysis buffer (100 mM
`Tris, pH 7.4, 500 mM sucrose, 0.5 mM EDTA, 0.1 mg/ml lysozyme, 1
`Ml/ml benzonase, and protease inhibitors) at 4 ml/g cell pellet. All
`manipulations were carried out at 4 °C. The lysate was stirred for 30 min
`and centrifuged (5000 X g, 20 min), and the pellet was resuspended in
`200 ml of Buffer A (400 mM KPi, pH 7.4, 20% glycerol, and 10 mM
`
`9'E0[l.IAAOG
`
`
`
`
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`112/§lio'oq['mA\m//:d11q(1101;p
`
`Early Molecular Modeling of 2D6——The initial sequence alignment
`was carried out using PSLBLAST (33) against a nonredundant protein
`data base comprising over 400,000 sequences. A resultant 1200—se~
`quence multiple sequence alignment was refined using the HMMER
`sequence profile alignment tools (34), and the 2D6 model was built
`using the homology modeling tools in ICM (35).
`(generation oj2D6 1 riincates— he 2136 truncates were generated by
`and left to stir for 30 min before being processed twice through a high
`PCR, introducing an Xbal site at the C terminus and an Ndel site at the
`pressure (10,000 psi.) disruption system (Constant Systems Ltd,
`N terminus plus various amino acid alterations. The N~terminal prim—
`Northants, UK). The lysate was centrifuged (100,000 X g, 1 h), and the
`ers were designed to remove the extreme N terminus, containing the
`supernatant was retained.
`putative membrane spanning region, and polyhistidine tags were added
`Protein Piirificcition—The protein was purified using an adaptation of
`at the N— or C terminus as described. After digestion with Ndel and
`a previously published method (38). All manipulations and purifications
`Xbal, the PCR products were subcloned into the pC\X/ expression vec—
`were carried out at 4°C. Purification followed a three—step process:
`tor (36). The sequence of all constructs was confirmed by automated
`Ni2+—nitrilotriacetic acid (Qiagen, Crawley, UK), hydroxyapatite H
`dideoxy—DNA sequencing.
`
`
`(j€fl6F6ltl0I’l
`of 2D67VIittcints—Niutations at residues Leu—230 an
`UK). Experiments showed that the protein was 95% pure after the first
`Leu—231 were introduced using the QuikChange mutagenesis kit (Strat—
`two stages. All purifications were carried out using an AKTA Purifier
`agene, La Jolla, CA) according to the manufacturer's protocol. Multiple
`100 instrument (GE Healthcare). The cleared lysate was applied to the
`mutations were introduced in single PCRs using semirandom primers,
`nitrilotriacetic acid column in buffer A, washed (5 column volumes of
`with multiple bases inserted at exact positions during primer synthesis
`wash buffer 400 mM KP,, pH 7.4, 20% glycerol, 10 mM ,8— mercaptoeth—
`(eg. the primer CTGAATGCTCTCCCCGTCl}{lRl§lCTGCATATCC—
`anol, 50 mM glycine, and 100 mM NaCl) and eluted using a linear gradia
`CAGCGCTGGCTG was used to introduce Asn, Lys, His, Gln, Arg, and
`Ser at residue 230).
`ent of 0—100% Buffer B (400 mM KP,, pH 7.4, 20% glycerol, 10 mM
`[3—mercaptoethanol, and 100 mM EDTA) over 10 column volumes. The
`Expression of2D6 Trnncates and Mutants to Test Soli4bi'li'ty—lnitial
`_
`.2+
`.
`.
`.
`.
`.
`.
`.
`
`
`expression trials or all’2136 constructs were performed using the host
`Buffer C (50 mM KP,, pH 7.4, 20% glycerol, and 10 mM ,8—mercaptoeth—
`DH10B (lnvitrogen). Cultures (5—ml scale) were grown overnight at
`anol) and loaded onto the hydroxyapatite column. The column was
`37 “C in LB broth supplemented with ampicillin. These cultures were
`washed (5 column volumes of buffer C), and the protein was eluted
`used as a 1% inoculum in modified Terrific broth (100 ml), supple—
`using a linear gradient of 0—100% Buffer D (400 mM KP}, pH 7.4, 20%
`mented with ampicillin, 1 mM thiamine, 0.5 mM 5—aminolevulinic acid,
`glycerol, and 10 mM [3—mercaptoethanol) over 10 column volumes. The
`and trace element solution (37). When the cultures reached an A600 of
`P450—containing fractions (posthydroxyapatite) were pooled and con»
`0.6 at 30 "C, expression was induced by the addition ofisopropyl 1—thio—
`centrated using a 15 ml Amicon centrifugal filter device (Millipore
`[3— Dgalactopyranoside to a final concentration of 1 mM. The cells were
`Corp., \X/atford, UK) to ~15 ml. The concentrated sample was applied
`harvested after a further 16 h at 30 °C by centrifugation at 5000 >< gfor
`o
`[41
`10 min at 4 C.
`
`‘QJQQIIIQAONU0$OJ qqSUTIQQUTSUH/9309138759['BD"p9l.IIOlfi‘&'llS.I9AlUfl1Uq.I9pII'B
`
`
`
`Activity /ism)/s—Activity assays were performed in a 384~well micro»
`plate using an LIL Biosystems Analyst HT (Sunnyvale, CA). Excitation
`and emission wavelengths were set at 405 and 530 nm (with a 425—nm
`diachronic mirror). The assay was performed using adaptations of pre-
`viously published methods (40, 41). 0 -128 [.LI\/l 4~methylaminomethyl~
`7—methoxycoumarin (GlaxoSmithKline) was added to 10 [.LII’10l of 2D6
`in the presence of 10 mmol of cumene hydroxyperoxide (as an electron
`donor), 1 mM dithiothreitol, 40 mM Kifi, p. .
`.5, and 20% glycerol._Read—
`ings were taken every 60 s for 10 min. The 2D6 L230D/L231R metabo—
`
`MARCH 17, 2006-VOI Ul\/lF 781-NUMBFR l
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`glycerol, 100 mM NaCl, and 10 mM [3—mercaptoethanol). The resulting
`fractions were analyzed by SDS—PAGE (Cambrex, Nottingham, UK),
`peptide mass fingerprinting (l\/licromass, Manchester, UK), protein
`assay (Coomassie Plus; Pierce), and P450 content (CO difference assay).
`Carbon Monoxide Difiference Assozy—Concentrations of P450 were
`estimated spectrophotometrically using a Cary Bio 100 instrument
`(Varian, Crawley, UK) from the difference spectra determined for the
`formation of the carbon monoxide complex with the protein after
`V
`E‘, Sp€C11
`QC 1V1 YO
`
`SI/tbC6lll/tlaf” FfdClloflclllofl-—ESCl1€}’lCl’ll6Z coli pellets were resuspended
`in TSP, buffer (200 mM Tris acetate, pH 8.0, 500 mM sucrose,
`1 mM
`phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme) and incubated
`on ice for 1 h. Cell pellets were recovered by centrifugation at 5000 X g
`for 10 min at 4 °C and resuspended in lysis buffer (0.5 M KPi, pH 7.6, 20%
`glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol).
`Cells were lysed using sonication (four 30—s pulses with 5—min intervals
`at 50% of maximum power), and cell debris was pelleted by centrifuga
`tion at 5000
`gtor 10 min at 4 C. he supernatant was turther clarified
`by centrifugation at 100,000 X gfor 1 h at 4 °C in a Beckman TLNe100
`rotor. The resultant supernatant was used as the cytosolic fraction (con—
`taining “soluble" P450), and the pellet contained the ineinbrane fraction.
`Expression of2D6 at Large Sccile——A shake flask containing 100 ml of
`[B was inoculated with a single colony taken from a fresh transformant
`previously plated out onto LB agar. The flask was incubated at 30 °C
`with constant shaking at 120 rpm for 14 h before transfer as a 2% inoc-
`ulum to the final stage. The final stage consisted of2.5 liters ofMTB and
`1"o glycerol in a 3.6~liter laboratory scale termenter (ln'torsAG, bWltZ€t'—
`land). All media were supplemented with 100 /Jog/1’1’1l ampicillin. After
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`Cytochrome P450 2D6 Crystal Structure
`
`TABLE 1
`
`Value
`
`40-30 (3.l1—3.00)
`94, 1 1 1
`39,448
`90.0 (90.7)
`11.2 (1.6)
`0.107 (0.614)
`
`Crystallographic statistics
`Parameter
`Data collection statistics“
`Resolution (A)
`Observations
`Unique reflections
`Completeness (%)
`Average 1/0‘!
`R
`I;
`nierge
`Refinement statistics
`Resolution (A)
`R factor’
`RING (4% of data)
`Root mean square deviations from ideality
`0.016
`Bond lengths (A)
`1.65
`Bond angles (degrees)
`Average B factors (A )“
`56.9 (7240)
`Protein main chain
`57.5 (6996)
`Protein side chain
`52.9 (172)
`Hemes
`39.0 (11)
`Waters
`53.9 (10)
`Sulfates
`" Data for the highest resolution shell are given in parentheses.
`" Rniergc : E11; ” <1,v>1/3(1)
`"R = EHF01 * 1F.H/E1F..1'
`‘’ The number of model atoms is given in parentheses.
`
`40~3.0
`0.230
`0286
`
`lizes 4—methylaminomethy1—7—methoxycoumarin with a Km of 67.6 [.LM
`and a 1/max of 2.85 i 0.25 p.M/min.
`Crystallization and Data C0llecti0n——Crystals of the 2D6 L230D/
`L231R mutant construct were grown at room temperature ("V 20 °C) by
`free interface diffusion using Topaz XRAY chips (Fluidigm). A solution
`of the protein at 60 mg/ml in a buffer of 50 mM KPi, pH 7.4, 100 mM
`NaCl, 20% glycerol, and 5 mM ,8~mercaptoethano1 was loaded into the
`chip along with a range ofsolutions based on simple dilutions with water
`of a solution of 2.0 M ammonium sulfate, 0.1 M sodium citrate, pH 5.6,
`and 0.2 M potassium sodium tartrate, with the optimum dilution cen—
`tering around an ammonium sulfate concentration of 1.48 -1.52 M. Fol»
`lowing protein and reagent loading, the chip interface line was opened
`and allowed to remain open for the duration of the crystallization proc—
`ess. All chips utilized water as the hydration fluid to maintain the envi»
`ronment of the chip at ~100% relative humidity, and the chips were all
`prehydrated for at least 24 h prior to the experiment. Crystals typically
`appeared after a few days and continued to grow for a further 5-10 days,
`usually growing out of a globular gel~1ike aggregate, which generally
`termed atter about 24 h. The crystals are rectangular plates with dimen-
`sions that rarely exceed 80 X 20 X 10 um. The crystals were harvested
`from the chips and stored in a solution of 1.8 M ammonium sulfate, 0.1
`M sodium citrate, pH 5.6, 0.2 M potassium sodium tartrate, and 20%
`glycerol. The crystals were mounted directly from the harvesting solu—
`tion and flash—frozen in liquid nitrogen. X—ray diffraction data were
`collected with a MarMosaic 225 CCD detector (Mar Research) at 100 K
`at a wavelength of 0.9538 A using beam line 1D23—1 at the European
`Synchrotron Radiation Facility. Due to the small size of the crystals, it
`was necessary to use exposure times on the order of 10-20 s per 0.5
`oscillation, which resulted in considerable radiation damage during the
`data collection. The final native data set was assembled from 114 images
`collected from three crystals with refined mosaicities of 0.44, 0.51, and
`066°. The data were processed and scaled using the HKL2000 suite of
`programs (42). Structure factors were derived from the reflection inten—
`sities using the CCP4 suite 0 programs (43). The crystals belong to
`space group P21212 with unit cell dimensions a — 145.1 A, b — 155.5 A,
`C = 95.8
`Table 1 gives a summary of the data collection statistics.
`Structure Determinati0n—1he structure was solved by Molecular
`Replacement using PHASER (44) with a 2C9 crystal structure (Protein
`
`Data Bank code 1OG2) as a search model. The search model included all
`protein atoms for the 2C9 monomer from Pro—30 to Val~490 and
`excluded the heme group and water molecules. The sequence identity
`between the 2C9 and 2336 sequences was 40.7% in a protein sequence
`alignment c region, ri:iatc1=1i—n.g—18 ofA59 residues. A coin»
`vincing molecular replacement solution comprising four molecules in
`the asymmetric unit was found showing 222 symmetry (consistent with
`peaks observed in the self—rotation function). The resulting set of phases
`was used to calculate 2P0 - Fl and P0 — Fl (where P0 represents the
`observed structure factor and PC is the calculated structure factor) elec—
`tron density maps. The presence of large positive peaks in positions
`corresponding to the heme iron atoms confirmed that the molecular
`replacement solution was correct.
`1,),/,A,B.A,/,.
`Hg?
`
`,3)
`
`In N
`
`using multiple cycles of model building with the molecular graphics
`program COOT (45), followed by structure refinement with RFFMAC
`(46). Due to the limited resolution of the diffraction data, very tight
`noncrystallographic symmetry restraints were imposed throughout the
`refinement. 1n the last cycle, these restraints were relaxed for a small
`number of protein residues, which appeared to exhibit some differences
`in conformation between the individual molecules. The last cycle of
`refinement also incorporated TLS parameters (47) to model anisotropic
`
`
`
`1.,J19/§ii0'oq['mA\m//:d11q(1101;p91201uAAO([
`
`
`
`
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`‘QJQQLIIQAONU0S9111?SII1l99tI13iIT_.[/93119138759['BD’p91.IIO1fi‘.KJ1S19A1Uf)_1]1q.I9pt1"B
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`
`
`
`
`resulted in significantly better R and Rfme values (0230 and 0.286,
`respectively) compared with an equivalent non—TLS refinement cycle,
`where R and Rhee values were 0.253 and 0.311, respectively. The final
`refinement statistics are given in Table 1. The final model contains
`14,429 atoms and comprises four 2D6 molecules, two sulfate ions,
`closely involved in crystal contacts, and 11 water molecules. The protein
`model includes the residues 52~ 497 (full—length protein sequence num—
`bering) and additionally a short stretch of the proliiie—rich N—terminal
`
`built only as alanines, since the electron density maps were insufficiently
`clear to unambiguously assign the correct residue side chains. In a Ram—
`achandran plot, 84.7% of residues are in the most favored regions as
`defined by PROCHECK (48), with 12.9 and 1.6% of residues in the addi—
`tionally allowed and generously allowed regions, respectively, and 0.8%
`in disallowed regions. The four protein molecules are related by 222
`symmetry and are all essentially the same with only minor differences,
`the root mean square deviations between Ca atoms for all possible
`
`H ' , . ,
`
`
`
`A/D, 0.14 A, B/C, 0.12 A; B/D, 0.13 A, and C/D, 0.14 A. The protein
`figures were drawn using PYMOL (49).
`Recent Modeling 0f2D6—A11 molecular dynamics simulations men»
`tioned here were performed with the CHARMm program (50) on a
`Silicon Graphics 48xR12000 processor Origin server. Visualizations
`were carried out with a Silicon Graphics Octane work station using the
`QUANTA program (51). The debrisoquine ligand was built, and ab
`initio charges (3—21G" natural atomic orbital) were calculated using the
`
` '' ' pro ram 52 . Substrate doc <ings were carried out man ua y
`
`by placing the compound in a number of plausible starting poses and
`then minimizing them in the protein using CH/\RMm, with 500~step
`Steepest Descent followed by 5000—step Adopted Basis Newton Raph—
`son. A distance constraint was used to keep the iron—oxygen atom
`within reacting distance to the site of metabolism. For the heme group,
`optimization of a “picket fence" porphyrin, containing an iron—bound
`oxygen atom on the distal side and a thiomethyl group on the proximal
`side, was carried out at the unrestricted l-lartree—l3ock level, using a
`6—31L::"‘ basis set. he charges used were natural atomic orbital charges.
`The iron—cysteine bond was formed in CHARMm using a patch RTF file
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`written according to the standard Cl-{ARMm protocol. The heme
`model was least—squares—fitted to the crystal structure but otherwise
`was kept rigid during the simulation, thus alleviating the need for special
`parameters for the octahedral iron complex, which is not readily han—
`1] H
`.
`1
`5
`HQ
`
`RESULTS AND DISCUSSION
`
`Cytochrome P450 2D6 Crystal Structure
`
`cient to obtain 100% soluble P450, so it was decided that mutation of
`
`
`
`112/§¥1o'oq['mA\m//:d11q(1101;p9oeo1u.iAo([
`
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`E
`§_
`5
`
`hydrophobic surface residues on ZD6 would be required to obtain
`increased solubility, using truncate 5 as a template.
`Generation of ZD6 Mutants with Improved Sol:/ibility—A model of
`'? —%$wnce$ignmefiQ
`crystal structure of bacterial cytochrome P450 BM3. The sequence
`identity of ZD6 to BM3 is low, and although some regions of the ZD6
`Expression of2D6 Truncates———————P450 ZD6 expresses at high levels in
`model can be considered reliable, the quality of the ZD6 model around
`E. coli if the extreme N terminus containing the putative membrane—
`the putative substrate access channel is less good. However, careful
`spanning region is replaced with different signal sequences, the OmpA
`analysis of the model revealed a patch of hydrophobic residues in the
`signal sequence (53), or the sequence used to express P450 17oz in E. coli
`loop region between the F and G helices (Fig. 2), which was proposed to
`(54). However, ZD6 protein generated with these N—terminal sequences
`be situated on the surface ofthe protein and could possibly contribute to
`associates with E. coli membranes and is not amenable to crystallization.
`protein aggregation and membrane association. Two ofthese residues,
`
`formed by removal of this hydrophobic N—terminal sequence (e.g. 2C3
`idues using primers randomized at specific nucleotides. Fifty—two
`(38), 2C5 (25), 2C8 (28), 2C9 (26, 27), and 3A4 (29, 30)). 2C3, unlike
`mutants of ZD6 were generated (Fig. 3A) and tested for expression of
`many other P450s, contains several hydrophilic residues between the
`holo—P450. The solubility of those mutants expressing holo—P450 was
`membrane—spanning region and the PPGP motif, which is required for
`then tested by partial purification in high salt buffers (Fig. 3B). Six of the
`heme incorporation (55), and a simple truncation was sufficient to
`mutants showing the highest solubility and expression of holo—P45O
`obtain almost 100% cytoplasmic expression of 2C3 (38) (2C3d; see Fig.
`were selected for larger scale growth, further purification, and crystalli—
`1). Soluble expression of 2C5 required the introduction of residues 2- 6
`Zation trials (L230T/L231K, L230D/L231R, L230A/LZ31S, L230N/
`of 2C3d to obtain similar levels of soluble protein (25). A simple trun—
`.s231D, L230T/L231D, and L23ON/L231R). Of these, ZD6 L230D/
`,
`F
`. '
`' .
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`see Fig. 1). Since the sequence preceding the PPGP motifis quite hydro-
`was used in an Subscqucnt Studies‘
`Crystal Structure of ZD6 Shows
`phobic in ZD6, additional truncates were generated (truncates 2—7; see
`Crystal Sn.MCtW.e 0f2D6_The 30
`F18 D7 which Contained fusions between the N termini of 2C3d and 3
`the characteristic P450 fold as seen in other members of the family (Figs.
`Variety of different residues in the N terminus of 2D6' Histidiiie tags
`4 and 5) The lengths and orientations ofthe individual secondary struc—
`were used both N— and C—terminally. The most successful expression of
`tum} elements in QD6 are Very Similar to those Seen in 2C9 (Supp1emen_
`ZD6 was obtained using truncate 5, but this truncation was not suffi—
`ta} Fig‘ 1)‘ A Structural alignment of ZD6 with 2C9 using the program
`LSQMAN (56) gave a root mean square distance of 1.16 A for 389
`aligned Ca atoms (an equivalent alignment with 3A4 gave a root mean
`I
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`between ZD6 and 2C9, there are six main areas where significant differ»
`C
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`egieis an e. eel“ ‘ZDZO :1
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`resulting in a shorter ioop between it and helix D (the differences span—
`ning residues 139-148). Although the total number of residues is the
`same as in 2C9, this short loop substantially reduces the interactions
`between the C—D connection region and the G—H loop in ZD6 (as evi»
`denced by the observed differences between the C-}—H loop conforma—
`
`
`
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`2:23 FL
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`2cS FL
`2C9 FL
`2:36 FL
`2:313 ‘I’ rum: 1
`206 Truncz
`206 TrunC3
`286 Truncs:
`286 Trunc ‘S
`
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`296 Trunc?
`
`FIGURE 1. N-terminal sequence alignment of the 2D6 truncates with other P450
`isozymes. The full-length (FL) N-terminal sequences of 2C3, 2C5, 2C9, and 2D6 are
`shown with the soluble 2C3d sequence and the ZD6 truncates. Trunc1,ZD6 wi|d—type
`sequence, truncated at residue 23 to remove the membrane—spanning region, with
`N—terminal Hiss tag. Trunc2, 2D6 truncated at residue 34, with Hiss tag and residues 2—1 0
`of 2C3d inserted at the N terminus. Trunc3, 2D6 truncated at residue 25, with N—termina|
`Hiss tag. Trunc4, ZD6 truncated at residue 34, with Hiss tag and residues 2432 of 2E1
`inserted at the N terminus. Trunc5, 2D6 truncated at residue 34, with C-terminal His), tag
`and residues 2~10 of 2C3d inserted at the N terminus. Trunrfi, ZD6 truncated at residue
`25, with C-terminal His,‘ tag. Trunc7, 2D6 truncated at resid ue 32, with C-terminal His4 tag
`and residues 2—6 of 2C3d inserted at the N terminus.
`
`of [3 sheet 2 (residues 380 -392), where there is a considerable shift in
`position ofthe two strands relative to sheet 1. In ZD6, these strands bend
`up toward the underside of sheet 1 much more than in the case of ZC9,
`this closer packing between sheets being facilitated by 2D6 having more
`small hydrophobic side chains than 2C9 in this interface.
`The other four areas showing large differences between ZD6 and 2C9 are
`situated on the distal face of the protein. Three of them are directly involved
`in defining the shape and character of the protein's active site. The most
`obvious difference is the position of the ‘r helix and'fhe i-—G loop. Klthough
`there are substantial differences between the F—G loops in the literature 2C9
`
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`
`
`modeling.The secondary structure of BM3 is shown below the al ignment, with the predicted ZD6 structural elements shown above.Also included is the rabbit ZC5 sequence, (all three
`sequences are wild-type). The 2D6 residues Leu-230 and Leu-231 are highlighted.
`
`MARCH 17, 2006-VOI Ul\/lF 781-NlJl\/lBFR ll
`
`« 3" :3
`
`/OUR/\/N OF B/O/OG/CM CHF/l/IISTRY 7617
`
`Vanda Exhibit 2024 - Page 4
`
`VNDA 02699734
`
`Vanda Exhibit 2024 - Page 4
`
`

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`Cytochrome P450 ZD6 Crystal Structure
`
`FIGURE 3. Expression and solubility studies of
`the 2D6 mutants. A, the 52 L230/L231 double
`mutants. The mutants are marked (X). B, expres-
`sion levels and solubility. The expression levels are
`represented by the bars (scale on the /eftaxis), and
`the solubility is shown by the line plot (scale on the
`right). The six mutants selected for large scale
`study are highlighted with arrows.
`
`B
`
`gm.
`
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`structures (Protein Data Bank codes 1()C-)2 and 10GB versus 1R90), the
`situation with ZD6 is clearly different from 2C9. The F helix in 2D6 has two
`additional turns and arcs down much more closely over the heme pocket
`toward the N—terminal end of strand 2 of [3 sheet 1. This difference in the
`length of the F helix correlates strongly with an observed shift in the posi~
`tion ot strands 1 and 2 ot [3 sheet 1 (trom resi ese adopt a
`very different conformation from that seen in 2C9. At the end of the F helix,
`the PG loop lies across the side of the B’ helix, thereby enclosing the side
`that is completelyopen in 2C9. The FAG loop then rejoins the G helix, which
`adopts approximately the same orientation as that of 2C9, except that there
`is a significant shift along the helical axis, such that the turns do not align
`with each other. The quality of the electron density maps for the F—G loop
`region were unfortunately not high enough to give a completely satisfactory
`model for this important part of the structure, and for this reason, only a
`polyalanine trace was built tor part ot this loop. there is no sign ot an 1-
`helix, although the backbone of a short G’ helix does seem to be present.
`The two remaining differences between the ZD6 and 2C9 structures are
`also related to the F helix shift. The B’ helix in ZD6 is pushed out away from
`the heme pocket, and there are an additional three residues in the loop
`immediately following it (residues 101-118). Similarly, on the opposite side
`of the F helix from the B’ helix, [3 sheet 4 (residues 468 — 487) adopts a shift
`in confo