A Three-Dimensional Protein Model for Human Cytochrome P450 2D6

Chem. Res. Toxicol. 1996, 9, 1079-1091

1079

A Three-Dimensional Protein Model for Human Cytochrome P450 2D6 Based on the Crystal Structures of P450 101, P450 102, and P450 108 Marcel J. de Groot,†,‡ Nico P. E. Vermeulen,*,† Jeroen D. Kramer,†,‡ Fre´de´rique A. A. van Acker,†,‡ and Gabrie¨lle M. Donne´-Op den Kelder†,‡ Leiden/Amsterdam Center for Drug Research (LACDR), Divisions of Molecular Toxicology and Medicinal Chemistry, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands Received January 2, 1996X

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 Å away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a threedimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Introduction Cytochromes P450 (P450)1 constitute a large superfamily of heme-containing enzymes, capable of oxidizing and reducing a variety of substrates, both of endogenous and exogenous origin. Cytochrome P450 2D6 (P450 2D6 (1)) is a polymorphic member of the P450 superfamily and is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. Several inactivating alleles have been reported (2-4). This results in a deficiency in drug oxidation known as the debrisoquine/sparteine polymorphism, which affects the metabolism of numerous drugs. Poor metabolizers, which have two nonfunctional P450 2D6 alleles, show a diminished metabolism of these drugs compared to extensive metabolizers which have at least one functional allele. Today, various antiarrhythmics, β-adrenoceptor antagonists, antidepressants, opiates, and neuroleptics are known to be substrates for human P450 2D6 (5-7). These compounds represent a variety of chemical structures, common characteristics being the presence of at least one basic nitrogen atom, a distance of 5 or 7 Å between the oxidation site and the basic nitrogen atom, * To whom correspondence should be addressed. † Division of Molecular Toxicology. ‡ Division of Medicinal Chemistry. X Abstract published in Advance ACS Abstracts, September 1, 1996. 1 Abbreviations: P450 102, cytochrome P450 BM3/102; P450 101, cytochrome P450cam/101; FAD, flavin adenine dinucleotide; GBR12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine; PDB, Brookhaven Protein Data Bank; P450, cytochrome P450; SRS, substrate recognition region; P450 108, cytochrome P450terp/108.

S0893-228x(96)00003-3 CCC: $12.00

a flat hydrophobic area close to the site of oxidation, and negative molecular electrostatic potential values above the planar part of the molecule (8, 9). Theoretical models predicting the possible involvement of P450 2D6 in the metabolism of a potential drug are important for evaluating the risks possibly associated with marketing drugs (especially when their metabolism depends to a high extent on P450 2D6). Several small molecule models have been reported using a variety of substrates or inhibitors (8-12). In order to determine the nature and the properties of the protein sites responsible for the characteristics of these models, however, a three-dimensional representation of the entire active site of P450 2D6 is required. Subsequently, the protein interaction sites, which can be determined from the protein model, can be implemented into small molecule models, such as previously determined at our laboratory (8, 13), in order to further enhance their predictive value. As yet, crystal structures have been resolved for only four bacterial, soluble P450s: P450 101 (P450cam) (14), P450 102 (P450BM3) (15, 16), P450 107A (P450eryF) (17, 18), and P450 108 (P450terp) (19). The crystal structure of P450 107A (18) shows some significant differences compared to the crystal structures of P450 101, P450 102, and P450 108, especially in the regions of the A-, B′-, and F-helices (18). The coordinates of the P450 107A crystal structure (17, 18), however, have not been published yet. Furthermore, crystals have been reported for the first soluble eukaryotic P450, P450 55 (P450nor) from Fusar© 1996 American Chemical Society

1080 Chem. Res. Toxicol., Vol. 9, No. 7, 1996

ium oxysporum, although no crystal structure has been reported as yet (20). Homology modeling has been used to develop structures of P450s for which sequence information is available, but X-ray structures are lacking. These models have to be verified by either crystallization or site-directed mutagenesis experiments (21). The increasing number of crystal structures, however, should facilitate the homology modeling efforts (21). In literature, several homology models have meanwhile been reported for (complete and partial structures of) eukaryotic P450s based on the crystal structure of P450 101 alone, namely: P450 1A1 (22, 23), P450 2B1 (24), P450 2C9 (6), P450 2D6 (25), P450 3A4 (P450NF) (26), P450 11A (P450scc) (27), P450 17 (P45017R) (28, 29), P450 19A1 (P450arom) (30, 31), P450 51 (P45014R) (32, 33), P450 105A1 (P450SU1) (34), P450 105B1 (450SU2) (34). Also a set of active site models for 4 different eukaryotic P450 isoenzymes have been reported (P450s from family 1, 2B, 3, and 19 (35)). Furthermore, the crystal structure of P450 102 has been used as a basis for homology models of a number of eukaryotic P450s, namely: P450s 2A6, 2B1, 2B4, 2C3, 2C3v, 2C9, 2D1, 2D6, 4A4, 4A11 (36), and P450s 2A1, 2A4, 2A5, 2A6 (37). A recent homology building study on human thromboxane A2 synthase (P450 5) using both P450 101 and P450 102 as templates indicated that it may be necessary to carefully reexamine all previous models based on P450 101 alone with the aid of the recently elucidated crystal structures (38). Some protein models have very recently been constructed using the three available crystal structures of bacterial P450s: P450 2B1 (39) and P450 19 (P450arom) (40, 41). P450 102 is considered to provide the most useful structural information for homology studies on eukaryotic P450s, since this well-characterized and crystallized bacterial enzyme belongs to the so-called class II P450s (15) to which many eukaryotic P450s belong, as well. Class II P450s are bound to the endoplasmic reticulum and interact directly with a cytochrome P450 reductase, containing flavin adenine dinucleotide (FAD) and flavin mononucleotide, while class I P450s are found in the mitochondrial membranes of eukaryotes and in most bacteria and require an FAD-containing reductase and an iron-sulfur protein (putidaredoxin) (15). The aim of the present study is to derive a threedimensional model for P450 2D6 and more specifically for its active site based on a structural comparison of the crystal structures of P450 101, P450 102, and P450 108, in order to further explain and examine the substrate specificity of this polymorphic P450 isoenzyme. The results from the protein model will be used to extend the current substrate model of P450 2D6 (8, 13) with the complementary interaction sites between P450 2D6 substrates and P450 2D6 active site residues.

Computational Methods The coordinates of the crystal structures of P450 101 (14), P450 102 (15), and P450 108 (19) were retrieved from the Brookhaven Protein Data Bank (PDB) (42, 43) (reference codes 2CPP, 2HPD, and 1CPT, respectively). Recently, another (independently determined) crystal structure of P450 102 became available, which is very similar to the original crystal structure (16). The sequences of 66 known isoenzymes of the P450 2 family were retrieved from the Swiss-Protein Database (for reference codes see Figure S1, available as Supporting Information (original references for the used sequences can be found in a summary on the P450 superfamily (1))). For some of the alignments Clustal V (part of the CAMMSA package (44,

de Groot et al. 45)) was used. Homology building was carried out with Quanta version 4.0 (46) implemented on a Silicon Graphics Personal Iris workstation and IBM RS6000 workstations. Geometry optimizations were carried out using CHARMm version 22.0 (47, 48) on IBM RS6000 workstations. For substrate and inhibitor docking into the protein model Quanta and CHARMm were used as well. SETOR (49, 50) (on a Silicon Graphics Personal Iris) was used for visualization. Alignment. A multiple alignment was generated containing 66 members of the P450 2 family (Supporting Information, Figure S1) using Clustal V (44, 45). The sequences of P450 101, P450 102, and P450 108 were aligned based on structural elements present in the crystal structures, a so-called “structural alignment” (40, 51). Within this procedure, sequences are aligned based on a structural superposition of the structures involved. We have chosen for this structural comparison in order to derive an alignment for the three bacterial enzymes and not for one of the several available automated sequence alignment procedures, since structure conservation often seems to be more important than sequence conservation (51). The resulting alignment was subsequently aligned with the results from the multialignment of the P450 2 family. The latter procedure was mainly carried out manually, since automated procedures have been shown to align eukaryotic sequences incorrectly with bacterial P450s (51). As already indicated in the literature (51), the resulting alignment cannot be absolutely correct in the absence of structural or other information for any of the eukaryotic P450s. The core region, containing the I-helix, the L-helix, and the heme coordination region, of all available crystal structures is very similar (18, 21, 40, 51), indicating the three-dimensional structure of these P450s is maintained despite a low sequence homology, while other regions (e.g., the active site region containing the B′-helix (18, 21)) are less similar (18, 21, 40, 51). For this reason, the core region of a homology model of a P450 based on these crystal structures will likely be a reliable representation, while other parts will remain speculative. Various types of information were therefore used to eventually derive the alignment as presented in Figure 1 for P450 101, P450 102, P450 108, and P450 2D6, namely: highly conserved amino acids and domains, a comparison of predicted and observed secondary structure elements, and mutation data on various members of the P450 2 family. Highly Conserved Residues and Domains. From various alignments between different P450s and P450 101, it has become evident that P450s contain several well conserved domains. These include the oxygen binding domain located around the I-helix and the heme-binding domain, containing the fifth (axial) ligand of the heme moiety (Cys357 in P450 101). Another conserved region, which is present in eukaryotic P450s, in P450 102 and P450 108 but absent in P450 101, is an aromatic region containing an A1-X-X-P-X-X-A2-X-P-X-B-A3 sequence (see Figure 1, residues 409-420 in P450 2D6), with Ai ) F, W, or Y, B ) R or H, and X ) a weakly conserved amino acid (6, 35, 52-54). A phenylalanine has been indicated to be important for binding the electron donor to P450 101 (Phe350 (55), corresponding to Phe393 in P450 102 and Phe370 in P450 108). Also several positively charged amino acids in P450 101 were indicated to play a role in binding the electron donor (Arg72, Arg112, Lys344, and Arg364 (55)). The negatively charged propionyl groups of the heme moiety interact with a small number of amino acids which are conserved in several membrane-bound P450s: Arg112, Arg299, and His355 in P450 101, Trp96, His100, and Arg398 in P450 102, and His110, Arg114, Arg319, and His375 in P450 108 (Figure 1). The above-mentioned amino acids have been important for determining the final alignment as presented in Figure 1 and will be discussed further in the sections concerning the separate structural elements of P450 2D6. Secondary Structure Elements. The (predicted) secondary structure elements of several P450s have been compared with those of P450 101. The results indicate that the C-, D-, G-, I-, J-, K-, and L-helices are probably conserved for membranebound P450s (53, 54). Further, it has been established that

Protein Model for P450 2D6

Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1081

Figure 1. Alignment of P450 2D6 with P450 101, P450 102, and P450 108. SRS regions according to Gotoh (54) have been indicated. The conserved WXXXR sequence at the amino terminus of the C-helix (51) and the conserved EXXR region at the C-terminal end of the K-helix (35, 40, 51) are indicated. An aromatic region containing an A1-X-X-P-X-X-A2-X-P-X-B-A3 sequence, with Ai ) F, W, or Y, B ) R or H, and X ) a weakly conserved amino acid (6, 35, 52-54), is labeled “aromatic” (see text). Asterisks indicate the conserved region F(G/S)XGX(H/R)XCXGXX(I/L/F)A (40). amino acids exhibit certain preferences for the different types of secondary structure (36, 56-58). These data were incorporated manually in the alignment procedures. Mutation Studies on the P450 2 Family. The multialignment constructed for 66 known sequences of the P450 2 family

(Supporting Information, Figure S1) indicates which amino acids in P450 2D6 correspond to the proposed active site residues of other members of the P450 2 family. These data were then used to align the active site areas of the crystal structures with the presumed active site area of P450 2D6. After


de Groot et al.

Table 1. Structural Elements Present in the Derived P450 2D6 Protein Model, Template Choice, Quality of the Alignment, and RMS Deviations Relative to Template structure element

start

end

template

qualitya

RMS (Å)

β1-sheet B-helix N-terminal part of B′-helixb C-terminal part of B′-helixc C-helix F-helix G-helix β2-sheet I-helix J-helix J′-helix K-helix β3-sheet β4-sheet heme-binding domain L-helix β5-sheet

Gly66 Gly83 Arg101 Ile109 Pro126 Leu205 Pro230 Pro286 Asp292 Pro325 Val342 Pro354 Leu372 Gly392 Ser400 Gly445 Ser465

Asn82 Asp100 Asn108 Gly125 Lys146 Val229 Asp263 Asn291 His324 Asn341 Met353 Pro371 Lys391 Leu399 Leu444 Phe464 Arg497

P450 108 P450 101 P450 101 P450 108 P450 108 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102 P450 102

fair fair poord poord good poor poor fair very good very good fair good fair good very good very good poor

1.3 2.3 2.4e 2.4e 1.6 1.7 1.5 1.4f 1.4f 1.6 1.4g 1.4g 1.8h 1.8h 1.8 1.2 1.9

a Designations are relative to each other and based upon (a) sequence homology, (b) relative amount of experimental data used in the separate structural elements, (c) structural conservation of the elements between P450 101, P450 102, and P450 108, and (d) RMS values between template and P450 2D6 structural element. b Including the strand leading to the B′-helix. c Including the strand running from the B′-helix. d Since both P450 101 and P450 108 were used as templates for the B′-helix (see text), this structural element actually resembles a random coil structure rather than a helix. e-h RMS averaged over two structural elements or two parts of one element.

visual inspection of the P450 2D6 model, the alignment was adjusted slightly to orientate important amino acids of P450 2D6 toward the interior of the active site. Homology Building. Only those regions of the P450 2D6 enzyme surrounding the active site and responsible for binding molecular oxygen, the heme moiety, and the various substrates were incorporated into the model. The final P450 2D6 model consists of four segments: (1) the B-, B′-, and C-helices and the β1-sheet (Gly66-Lys146); (2) the F- and G-helices (Leu205-Asp263); (3) the I-, J-, J′-, K-, and L-helices, the β2-, β3-, β4-, and β5sheets, and the heme binding domain (Pro286-Arg497); and (4) the heme moiety. Based upon a thorough investigation of the sequence alignment results (Figure 1) and the structural superposition of the bacterial P450s, the template for homology building, i.e., either P450 101, P450 102, or P450 108, was selected for each structural element separately (see Table 1). Selection occurred on the basis of highest homology and proposed structural similarity (Class I or Class II). The backbone coordinates of the structural element of the respective template protein and the coordinates of identical side chains were copied to the P450 2D6 model. The heme moiety of P450 108 was incorporated into the P450 2D6 model. The N-terminal amino acid of segments 1, 2, and 3 was positively charged while the C-terminal amino acid of these segments was negatively charged. The coordinates of nonidentical side chains were calculated with CHARMm using standard atomic distances and bond angles (48). CHARMm was also used to close the gap between Asn108 and Ile109 which resulted from using different crystal structures as a template for two parts of the B′-helix. After a search for close contacts (interresidue distances smaller than 1.5 Å (48)) in the P450 2D6 model, these were removed in a conformational search procedure which minimizes the number of close contacts through side chain rotations in steps of 30°, with a cutoff distance of 3.0 Å (i.e., interacting atoms more than 3.0 Å apart are not taken into account). Using Quanta/ CHARMm, the lysine and arginine residues are by default positively charged, the aspartic and glutamic acids are negatively charged, while the histidines are kept neutral (as measured at physiological pH for corresponding model compounds in the absence of a protein environment (59)). His376, however, was changed into a positively charged residue since this amino acid aligns with a positively charged amino acid in P450 101 and P450 108 (respectively Arg299 and Arg319) and can form a similar interaction as the latter two arginines with the heme propionate group. The heme moiety contains a total charge of -2 and has both propionyl groups on the proximal side. This results in a total charge of -4 for the P450 2D6

model, which was smoothed over carbons and nonpolar hydrogens (47). Geometry Optimizations and RMS Deviations. In general, geometry optimizations of the protein structures were performed with the CHARMm forcefield (48) using a six stage optimization procedure with increasing conformational freedom at each stage of the calculation: (1) the coordinates of all nonhydrogens were fixed, (2) only backbone atoms and the heme moiety were fixed, (3 and 4) a harmonic constraint of 100 kcal‚mol-1‚Å-1 and 10 kcal‚mol-1‚Å-1, respectively, was applied to the backbone atoms, while the heme moiety and the CR atoms of the (six) terminal amino acids were still fixed, (5) only the heme moiety and the CR atoms of the terminal amino acids were fixed, and (6) only CR atoms of the terminal amino acids were fixed. The close contacts in the structure were removed in phase 2 of the optimization. At each stage of the optimization process, the steepest descend method (60) followed by the conjugate gradient method (60, 61) was used. All calculations were done with the “all hydrogens” definition of CHARMm (i.e., all hydrogens were explicitly present in the calculations (48)), while hydrogen bonds were explicitly considered in the calculations.2 In general, root mean square (RMS) deviations in the atomic positions were used to evaluate structural differences. Docking of Substrates and Inhibitors in the P450 2D6 Protein Model. Four compounds (Figure 2) were docked into the P450 2D6 protein model for various reasons. Debrisoquine (1) and dextromethorphan (2) are both marker substrates for P450 2D6 and template molecules for the P450 2D6 substrate model previously derived by Koymans et al. (8). Ajmalicine (3) is a potent, relatively large and semirigid inhibitor of P450 2D6 (9), while GBR12909 (4) is one of the largest known flexible P450 2D6 substrates. The active site of the P450 2D6 model must at least be capable of accommodating these four compounds, assuming that the inhibitor 3 binds in a similar manner compared to the substrates 1, 2, and 4. Compound 4 exceeds the boundaries of the original P450 2D6 substrate model to a significant extent (8, 13): the protein model can possibly indicate in which direction extension of the substrate model is allowed for. 2 CHARMm parameters used for all calculations were: NSTE 5000, NPRI 25, TOLG 0.10, STEP 0.020, TOLS 0.00, TOLENR 0.00. The hydrogen bond parameters used were: ALL, ACCE, IHBFRQ 50, CTONHB 3.5, CTOFHB 4, CUTHB 4.5, CTONHA 50, CTOFHA 70, CUTHBA 90. Hydrogen bonds to sulfur atoms of cysteines were not taken into account since CHARMm 22.0 lacks parameters for hydrogen bonds involving these atoms. The nonbonded parameters used were: RDIE, EPS 4.0, INBFRQ 50, CTONNB 10.50, CTOFNB 11.50, CUTNB 12.00, VSWITCH, SWITCH.


Figure 2. Compounds docked in the active site of the protein model for P450 2D6. Sites of oxidation by P450 2D6 are indicated with solid arrows. The dashed arrow indicates a predicted3 site of oxidation derived from the P450 2D6 substrate model (8, 13). The compounds were docked in several conformations into the active site of the protein model using Quanta. For this purpose, a conformational analysis was performed on the compound, resulting in various orientations and conformations of the compound within the active site of the geometry optimized protein model. The basic nitrogen atom(s) of the compounds was (were) orientated within hydrogen bonding distance from Asp301, which was proven to be important in the catalytic activity of P450 2D6 (62); the site of oxidation of the substrates was orientated above the heme moiety in a similar way as the site of oxidation of camphor in the P450 101 crystal (14). Subsequently, the protein-ligand (substrate or inhibitor) complex was geometry optimized using a six stage optimization procedure (see above), in which in the first stage the ligand is fixed as well. The amino acids interacting with the ligand obviously depend on the sequence alignment used. Further, due to the presence of many local energy minima on the potential energy surface of the protein-ligand complex (26, 63), the optimized orientations of both substrates and inhibitors within the active site of the protein model will depend on the initial conformation of the complex. Therefore, all interactions seemingly present within the protein-ligand complexes have to be considered with care. Determination of Binding Energies. The binding energy (enthalpy term only) of a ligand to an enzyme, Ebind(ligand), is determined by comparing the energy of the geometry optimized protein, containing the ligand in the active site, with the separate energies of the geometry optimized protein and ligand molecule, i.e.: Ebind(ligand) ) E(protein + ligand)opt - E(protein)opt - E(ligand)opt. Ebind(ligand) will only be used to compare binding orientations of the same ligand in the active site in order to find the orientation with the most favorable binding energy for that specific ligand. Direct comparison of binding energies of different ligands, however, is not warranted.

Results and Discussion Alignment. The alignment derived for P450 101, P450 102, P450 108, and P450 2D6 is shown in Figure 1 and contains only those parts which were incorporated into the present protein model. The substrate recognition site (SRS) regions as proposed by Gotoh (54) have been indicated in Figure 1 as well. Based on the crystal structure of P450 101, these regions have been indicated to be involved in substrate binding (54). The present (independently derived) alignment differs only in some details from the alignment recently reported for P450 101, P450 102, and P450 108 by Hasemann et al. (51). In the β1-sheet Hasemann et al. (51) aligned Gly46 in P450 102 with Gly60 of P450 101, while we aligned with Gly61 of P450 101. Their alignment of the N-terminus of the B′-helix is different from ours for P450 102 only 3

M. J. de Groot, unpublished results.


(51), while the C-terminus is aligned identically for all three crystallized P450s in both studies. The amino acids known to interact with substrates are not part of the actual B′-helix but rather of the strands leading to and from the B′-helix (Phe87 and Tyr96 in P450 101 (64); Glu77, Ile78, Ile99, Ser101, and Thr103 in P450 108 (19)). The alignment of these strands leading to and from the B′helix are identical in both studies for P450 101 and P450 108, which we used as templates for the two separate parts of the B′-helix. Therefore, we expect to identify similar amino acids responsible for substrate binding using our alignment, compared to the alignment of Hasemann et al. (51). In the crystal structure of P450 107A the orientation of the B′-helix is perpendicular to the plane of the heme while in the crystal structures of P450 101, P450 102, and P450 108 the axis of the B′helix is in the same plane as the heme (18). This indicates that the position of the B′-helix is relatively poorly conserved even when comparing bacterial P450s. In the present alignment the F-helix of P450 108 is shifted 3 amino acids to the right relative to P450 101 and P450 102, when compared to the alignment from Hasemann et al. (51), while the G-helix of P450 101 is shifted 4 amino acids to the right relative to P450 102 and P450 108 (51). A final difference is observed in the β5-sheet, where the N-terminal part of P450 101 is shifted one amino acid to the left relative to P450 102 and P450 108. Overall, only the differences in the alignment of the F- and G-helices are expected to affect (binding of ligands to) the P450 2D6 homology model compared to a model built using the alignment of Hasemann et al. (51). This issue will be discussed in the paragraph on the comparison between the protein model and the substrate model of P450 2D6. The structural elements (R-helices and β-sheets) of the P450 2D6 model will be discussed separately below, including the results of mutation studies used to support the alignment (Figure 1). The overall three-dimensional structure of P450s is maintained despite a low sequence homology, as the core regions of the crystallized P450s are all very similar, while other (specific) regions (e.g., the active site region (18, 21)) are less similar (18, 21, 40, 51). The amino acids mentioned in the text refer to P450 2D6 unless stated otherwise. Table 1 shows the quality of the alignment, and the RMS between the selected template and the corresponding structural element in the final P450 2D6 protein model. β1-Sheet (Residues 66-82). The structure-based alignment of the β1-sheet region reveals a low homology between P450 101, P450 102, and P450 108. However, from the multialignment of the P450 2 family (Supporting Information, Figure S1, and Figure 1 for P450 2D6) it is evident that this part of the sequence is highly conserved within the P450 2 family. Gly66 initiates the turn from the A-helix to the β1-sheet, in agreement with the preference of glycine residues for the C-terminus of an R-helix or a reversed turn (36, 56-58). Within P450 101 and P450 108 this function is taken over by the well-known helix-breaker (36, 56-58) proline (Pro51 and Pro38, respectively). Ala74 is part of a reverse turn between two strands of the β1-sheet and corresponds to a highly conserved glycine residue in the P450 2 family (35); only occasionally an alanine is found (Supporting Information, Figure S1, and Figure 1). Also P450 101 and P450 108 contain a glycine at this position. Pro77 is highly conserved within the 2B, 2C, 2D, and 2G subfamilies (Supporting Information, Figure S1). In P450


108 also a proline is present at this position, while P450 101 and P450 102 both contain a glycine residue at the corresponding position (Gly61 and Gly46, respectively (Figure 1). B-Helix (Residues 83-100). This part of the protein is not considered to interact with the substrate directly (54). However, since a mutation of Arg72 to Asn72 in P450 101 resulted in a change in oxidation rate (65), the corresponding Arg88 in P450 2D6 might also be inferred to be important, possibly because it forms an ion pair with the respective electron donor. Only the last amino acid in this region, Asp100 (which is no longer part of the actual B-helix), is part of SRS1 as inferred by Gotoh (Figure 1 (54)). B′-Helix and Strands Leading to and from the B′Helix (Residues 101-125). As also observed by Hasemann et al., the B′-helices are the most variably positioned regular secondary structure elements among the three P450 crystal structures, with very different lengths and orientations, as well as with a very low sequence homology (51). The conformation of the strands leading to and from the B′-helix is, however, somewhat better conserved. The entire B′-helix is part of SRS1 as defined by Gotoh (54). However, the amino acids known to interact with substrates actually are not part of the B′helix but of the strands leading to and from the B′-helix: Phe87 and Tyr96 in P450 101 (64), Glu77, Ile78, Ile99, Ser101, and Thr103 in P450 108 (19), and residues 112-115 in P450 2C2 (66) (corresponding to Val119 to Ala122 in P450 2D6, Supporting Information, Figure S1). Therefore, attention was focused upon the optimal alignment of these strands, resulting in a gap in the middle of the B′helix (Figure 1). Since both P450 101 and P450 108 were used as templates for the two separate parts of the B′helix and the surrounding residues, this structural element actually resembles a random coil structure more than an R-helix. The resulting alignment of the strands is supported by the positions of several conserved proline and glycine residues. Residue 102 is conserved within the P450 2 family, as either a glycine or a proline, and it is a proline (residue 86) in P450 101. Pro105 is conserved within the 2C, 2D, 2E, 2F, and 2G subfamilies and in P450 101 (residue 89). Gly111 and Pro114 are only conserved within the 2D subfamily and are also present in P450 108 (residues 93 and 96, respectively (19)). Gly118 in P450 2D6 corresponds with Gly85 in P450 102 and is conserved within the P450 2 family (Supporting Information, Figure S1). Leu121 of P450 2D6 aligns with Thr103 of P450 108, which was indicated to interact with substrates (19). Gly125 is conserved within the P450 2 family (67) and corresponds with a proline in P450 101 and P450 108 (residues 105 and 107, respectively (Figure 1)). In the crystal structure of P450 107A (for which the coordinates are not yet available from the PDB (42, 43)) the orientation of the B′-helix is perpendicular to the plane of the heme, while in the crystal structures of P450 101, P450 102, and P450 108 the axis of the B′-helix is in the same plane as the heme (18). This rotation of the B′-helix in P450 107A is required for the enlargement of the active site (18). Therefore, the strands leading to and from the B′-helix in P450 107A cannot be superimposed on the comparable regions in the other crystal structures. This indicates that the position of the B′-helix is relatively poorly conserved even when comparing bacterial P450s, and that although based on P450 101, P450 102, and P450 108 a reasonable alignment can be obtained for the strands leading to and from the B′-helix as indicated

de Groot et al.

above, this region remains one of the lesser reliable parts of any homology model. C-Helix (Residues 126-146). The highly conserved WXXXR sequence as identified by Hasemann et al. in the alignment of P450 101, P450 102 (present as WXXXH), and P450 108 at the amino terminus of the C-helix (51) is also present in P450 2D6 (Figure 1: Trp128-Arg132) and other members of the P450 2 family (Supporting Information, Figure S1). Pro126 initiates a turn between the B′- and C-helices and is conserved in the 2D and 2E subfamilies. Trp128 in P450 2D6 interacts with one of the propionate groups of the heme moiety, in analogy with Trp96 in P450 102 (15, 36) and His110 in P450 108 (19). Also the positively charged Arg132 of P450 2D6 interacts with a heme propionate group, which corresponds to Arg112 in P450 101 (36, 64, 68), His100 in P450 102, and Arg114 in P450 108 (19). The positive charge of Lys146 is conserved within the P450 2 family, P450 101 (Lys126), P450 102 (Lys113, shifted one amino acid to the left in the alignment), and P450 108 (Lys128). F-Helix and G-Helix (Residues 205-229 and 230263, Respectively). The crystal structures of P450 101, P450 102, P450 107A, and P450 108 reveal considerable differences, both in length and in sequence, in the region in between the F- and G-helices. Therefore, these helices might easily be incorrectly aligned by 3-4 amino acids (one helix loop (51)). In P450 107A the F-helix is shortened by two residues compared to P450 101 and shifted away from the substrate binding site by approximately 2.5 Å, in order to accommodate the rotation of the B′-helix (18). Since the prediction of the borders between the F- and G-helices and the connecting loop is difficult, this region will be one of the least reliable parts of our P450 2D6 model. In case SRS2 and SRS3 are present, they will be located in the F- and G-helices, respectively (51, 54). A mutation study on P450 2B1 indicated Phe206 to be important for stereo- and regioselectivity of various P450 2B1 substrates (24, 69-71). Mutation of the corresponding Phe209 in P450 2A4 changed its specificity to that of P450 2A5 (72-75). Both observations support that the corresponding Leu213 in the F-helix of P450 2D6 is part of the active site. Within the alignment, as given in Figure 1, the positively charged amino acids are orientated toward the exterior of the protein. The amino acids His258 (neutral) and Arg259 (positively charged) in P450 2D6 are conserved in part of the P450 2 family as histidine and lysine/arginine residues, respectively (Supporting Information, Figure S1). β2-Sheet and I-Helix (Residues 286-291 and 292324, Respectively). The I-helix contains the oxygen binding domain and is highly conserved in all P450s. The P450 2D6 residues Asp301 and Ser304 (in the I-helix) are probably involved in substrate binding similar to the corresponding amino acids in both P450 101 (Leu244 and Val247 (68)) and P450 108 (Ala363 and Thr367 (19)) which were both proposed to be part of SRS4 (54). Asp301 has recently been proven to be important in the catalytic activity of P450 2D6, as mutagenesis studies changing this aspartic acid into a glutamic acid, an asparagine, or an alanine indicated these mutants to be active, less active, and nonactive, respectively (62), thus demonstrating the necessity of a negatively charged group for catalytic activity. Ser289 may be important as well, since the corresponding residue in P450 2B1 (Glu282) was found to be involved in catalytic activity of the latter P450 isoenzyme (76). Also Ile297 is suggested to be directed


toward the active site cavity, since mutation of the corresponding amino acid in P450 2B1 (Ile290 (24)) and P450 2B11 (Asp290 (77)) results in mutant enzymes with altered activities and substrate specificities. The highly conserved oxygen binding domain (A/G)GX(E/D)T (14, 22, 35, 40, 64) consists in P450 2D6 of Ala305, Gly306, Met307, Val308, and Thr309. The alanine-glycine pair results in a distortion of the I-helix (40). Thr309 in P450 2D6 might also be involved in substrate binding, since mutation of the corresponding amino acid in P450 2B1 (Thr302 (24, 70, 71)), in P450 2C2 (Thr301 (78)), and in P450 2E1 (Thr303 (79)) alters the observed stereo- or regioselectivities. The P450 2D6 residue Thr310 is probably not involved in substrate binding, since the corresponding amino acid in P450 2B1 can be mutated without changes in the observed stereo- or regioselectivity (24). J-Helix and J′-Helix (Residues 325-341 and 342353, Respectively). In between the J- and K-helices, P450 102 and P450 2D6 contain an additional helical region which is absent in P450 101 and P450 108. This insertion, named J′-helix, is characteristic for microsomal P450s (15). The N-terminus of the J-helix is characterized by a proline (Pro325), which is conserved in the P450 2 family and the bacterial P450s. K-Helix (Residues 354-371). The P450 2 family contains a conserved EXXR region at the C-terminal end of the K-helix (35, 40, 51) with an internal hydrogen bond between the glutamic acid and the arginine (Glu362 and Arg365 in P450 2D6 (Figure 1)). Site-specific mutation studies on P450 2C9 have indicated that Leu359 (corresponding to Gly367 in P450 2D6, Supporting Information, Figure S1) is important for substrate interactions (80). Similar experiments changing Val363 into P450 2B1 (24, 69-71) indicate a possible role for the corresponding Val370 in P450 2D6 (Supporting Information, Figure S1). The last two amino acids of the K-helix are part of SRS5 (54), the majority of SRS5 being located in the β3-sheet (Figure 1). β3- and β4-Sheets (Residues 372-391 and 392399, Respectively). The β3-sheet contains the major part of SRS5 (Figure 1 (54)). The alignment of the region containing the β3- and β4-sheets is difficult due to the variable lengths of these sheets in the different crystal structures (51) and their low homology, stressing the necessity of a structural alignment for this part of the protein. However, Val374 is most likely orientated toward the active site of P450 2D6, since two different cDNAs for P450 2D6, expressed in two different cell lines (81, 82), contain either a valine or a methionine at position 374 and display different stereo- and regioselectivities toward metoprolol (82, 83). The corresponding Val367 in P450 2B1 (70, 71) and Val367 in P450 2B4 (84) (Supporting Information, Figure S1) were also proven to be involved in substrate binding or catalytic activity. These data enhance the probability that these three valine residues are structurally aligned in space and are orientated toward the active sites of these isoenzymes. The multialignment also suggests Leu372 to be part of the active site of P450 2D6. Mutations of the corresponding amino acid in P450 2C3/2C3v (Thr/Ser364, Supporting Information, Figure S1) alter the capacity of this isoenzyme to catalyze 6β-hydroxylation of progesterone (85). Arg299 in P450 101 (14) and Arg319 in P450 108 (19) were shown to interact (directly or via a water molecule) with a propionate group of the heme moiety and therefore probably correspond functionally with the positively charged His376 in P450 2D6 (Figure 1). The positive


charge (His or Arg) is conserved in the P450 superfamily (35), in the majority of the P450 2 family as a histidine (Supporting Information, Figure S1). Met377 might also be involved in substrate binding as suggested by mutation studies on P450 2D1 (86, 87). Gly392 is well conserved in the bacterial P450s and the P450 2 family (Figure 1 and Supporting Information, Figure S1) and initiates the transition between the β3- and β4sheets. Heme-Binding Domain (Residues 400-444). The heme-binding domain containing the 310(1), 310(2), and 310(3) helices and the cysteine ligated to the heme is highly conserved, both structurally and in terms of sequence identity, in P450 101, P450 102, P450 108, and the P450 2 family. Phe436 (Figure 1) is highly conserved in the P450 2 family (Supporting Information, Figure S1) and bacterial P450s (Phe350 in P450 101 (35), Phe393 in P450 102, and Phe370 in P405 108 (Figure 1)) and is suspected to be involved in the electron transport process from the electron donor to the heme moiety (55). Gly439 (Figure 1) is also highly conserved (35) and influences the conformation of the heme-binding domain. The conserved positively charged amino acid at position 441 (Arg or His) is assumed to interact with the negative charge of one of the propionate groups of the heme moiety (Figure 1 (14, 19)). The fifth (axial) ligand of the heme is a conserved cysteine (Cys443, Figure 1 (35)). At position 444 (Figure 1) a hydrophobic residue (leucine or isoleucine) is conserved, which can form a hydrophobic interaction with the heme moiety. The latter two amino acids are part of the conserved consensus F(G/S)XGX(H/R)XCXGXX(I/L/F)A (residues 436-450 (40) (Figure 1)). L-Helix (Residues 445-464). The L-helix is a very well conserved helix. The initial part of this helix is also part of the earlier mentioned conserved consensus F(G/ S)XGX(H/R)XCXGXX(I/L/F)A (residues 436-450 (40) (Figure 1)). Gly445 in P450 2D6 is largely conserved in the P450 2 family (Supporting Information, Figure S1) and defines part of the heme binding pocket (35). Ala449, Arg450, Glu452, and Leu461 are also highly conserved (35). Arg450 (Arg364 in P450 101) probably interacts with the electron donor in P450 101 (55). β5-Sheet (Residues 465-497). Very little information supporting this part of the alignment is available. Mutation of Gly478 in P450 2B1 (corresponding to Leu484 in P450 2D6, which is part of SRS6 (54), Supporting Information, Figure S1) changes the stereoselectivity for 16-hydroxylation of androstenedione and testosterone (24, 69-71, 88-90), indicating the importance of this residue for catalytic activity. The corresponding Ile395 in P450 101 (14) has been shown to interact with the substrate (camphor). Homology Building and Geometry Optimizations. For the construction of the P450 2D6 protein model, a template was chosen for each structural element (Table 1). Due to an overall better alignment (based on structural alignment and sequence homology) between P450 2D6 and P450 102 compared to P450 101 and P450 108, P450 102 was mainly selected as template protein for P450 2D6. Furthermore, P450 102 contains some insertions characteristic of microsomal P450s (15). Some parts, however, were taken from either P450 101 or P450 108 (see Table 1) due to a better local alignment. The resulting P450 2D6 protein model was geometry optimized in a six stage procedure (see Computational Methods). The resulting RMS values between the protein model before and after geometry optimization were 1.7


Figure 3. Protein model for P450 2D6. R-Helices are indicated in red, β-sheets in green. The heme moiety is depicted in yellow. Helices and sheets are named according to the crystal structure of P450 102 (15).

Å overall, 1.6 Å for the non-hydrogen atoms, 1.3 Å for the backbone atoms, 0.6 Å for the heme moiety, and 1.2 Å for the CR carbons. The RMS values between the CR atoms in the core regions of the three bacterial P450 crystal structures were reported to range between 1.9 and 2.0 Å (51), while RMS values between two monomers of the crystallized dimeric P450 102 were 0.8 Å for all CR atoms (15) and up to 1.8 Å in the region of the F- and G-helices (15). The RMS values reported between the crystal structures of P450 101 and P450 102 were 2.8 Å for all CR atoms (91). The RMS values between these crystal structures indicate that our P450 2D6 model is within the boundaries defined by the bacterial crystal structures. In the final protein model for P450 2D6 (Figure 3), the I-helix is positioned over the pyrrole rings C and D of the heme moiety in the active site. Docking Substrates and Inhibitors. Three substrates and one inhibitor (Figure 2) were selected to be docked into the active site of the P450 2D6 protein model. The rationale for docking substrates and inhibitors into the P450 2D6 protein model was to investigate whether the active site of the protein model is able to accommodate these specific compounds and to identify possible substrate recognition regions. Furthermore, information would be obtained on how the previously reported substrate model (8, 13) can be extended with substrate recognition regions from the protein, in order to enhance the predictive value of the substrate model toward possible P450 2D6 metabolism. Debrisoquine (1) and dextromethorphan (2) are both marker substrates for P450 2D6 and template molecules for the P450 2D6 substrate model as derived by Koymans et al. (8). Debrisoquine (1) is classified as a 5 Å substrate and dextromethorphan (2) as a typical 7 Å substrate. Ajmalicine (3) is a relatively large, potent, semirigid inhibitor of P450 2D6 (9), which possibly can be used as a template for inhibitors3 within the substrate model for P450 2D6. GBR12909 (4) is one of the largest known P450 2D6 substrates. Both 3 and 4 exceed the boundaries of the P450 2D6 substrate model (8, 13) to a significant extent. The protein model for P450 2D6 can possibly indicate in which direction extension of the

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substrate model for P450 2D6 is allowed for. Since for the substrates and inhibitor various conformations and orientations within the active site are possible, several protein-ligand complex conformations were generated through a conformational search on each compound within the active site of the protein model. In all orientations, the basic nitrogen atom of the ligand was orientated within hydrogen bonding distance from the carboxylate group of Asp301. The energy optimized orientations, however, appeared to be close to the initial orientation (derived from the conformational analysis), due to the presence of several local energy minima on the potential energy surface of the protein-ligand complex (26, 63). The oxygen atom which is incorporated into the substrate during metabolism by P450 2D6 was not considered explicitly during the docking process. In the literature two possible positions for the iron-bound oxygen atom in P450 (model systems) have been described: one perpendicular to the plane of the heme moiety (ferryl oxygen intermediate) (92) and a second one with the oxygen atom bridged in between the iron atom and one of the four heme nitrogen atoms (93, 94). Within our P450 2D6 protein model for a Fe-N bridged oxygen atom, only the bridged position between pyrrole rings A and B is available due to steric hindrance caused by the I-helix, which is positioned over pyrrole rings C and D. In the case of a Fe-N bridged oxygen atom, the oxygen lone pairs would therefore be positioned above pyrrole rings A and B of the heme moiety, while in the case of an oxygen atom bound to the iron atom perpendicular to the plane of the heme, the position of the lone pairs would not be as rigidly set. The existence of only two separate groups of substrates (either 5 or 7 Å) can easily be explained using the Fe-N bridged oxygen atom hypothesis. In our protein model, however, both proposals for the position of the oxygen atom can be accommodated and it is not possible, based on the available data, to identify the most likely structure for the iron-oxygen intermediate. Ab initio calculations are in progress on iron-porphyrins with an S-methyl axial ligand and an oxygen atom in either the bridged or perpendicular position in order to further investigate both proposals.3 Debrisoquine (1, Figure 2). Several proteinsubstrate complexes containing different orientations of the 5 Å substrate 1, resulting in 4-hydroxylation (the P450 2D6 specific metabolic reaction (2, 5)), were generated and subsequently optimized. The conformation of 1 in the protein-substrate complex with the lowest energy (most optimal binding geometry) is shown in Figure 4a. The conformation of 1 is one of two low energy conformations, which differ only in the conformation of the ring (data not shown). The oxidation site of the substrate is located above pyrrole ring A of the heme moiety in an orientation leading to the stereoselective formation of the 4(S)-hydroxyl metabolite. The prochiral benzylic carbon atom was orientated above the heme moiety in a similar way as the site of oxidation of camphor in the P450 101 crystal (14). Substrate-protein interactions at a distance smaller than 5 Å were present between 1 and amino acids from SRS1, SRS2, SRS4 (Asp301), SRS5, and SRS6 (54) and the highly conserved part of the heme-binding domain (Figure 1). The conformation of 1 shown in Figure 4a is nearly identical to the presumed global minimum energy conformation of 1 optimized in the absence of a protein environment. The amino acids interacting with 1 were as follows: Asp301



Figure 4. Active site orientations for compounds 1 (Ebind(1) ) -23.3 kcal/mol) (a, top left), 2 (Ebind(2) ) -42.2 kcal/mol) (b, top right), 3 (Ebind(3) ) -32.9 kcal/mol) (c, middle left), 4 aromatic hydroxylation (Ebind(4) ) -42.1 kcal/mol) (d, middle right), and 4 benzylic hydroxylation (Ebind(4) ) -35.0 kcal/mol) (e, bottom left). Hydrogen atoms have been omitted for clarity. Carbon atoms are shown in white, oxygen atoms in red, nitrogen atoms in blue, and iron atoms in cyan. Sites of oxidation (Ox) and heme nitrogen atoms have been labeled.

(I-helix, SRS4), Val370 (K-helix, SRS5), and Leu484 (β5sheet, SRS6). Dextromethorphan (2, Figure 2). The selected protein-substrate complexes contained different orienta-

tions of the rigid 7 Å substrate 2, with its basic nitrogen atom orientated toward Asp301 and the site of oxidation located above pyrrole ring A of the heme moiety. However, after geometry optimization of the protein-sub-


strate complexes, the position of the site of oxidation changed while the position of the basic nitrogen atom did not. In the most favorable protein-substrate complex, the conformation of 2 had changed, so that the site of oxidation had moved to a position above pyrrole ring B (Figure 4b), while substrate-protein interactions at a distance smaller than 5 Å were present between 2 and several amino acids from SRS1, SRS2, SRS4 (Asp301), SRS5, and SRS6 (54) and the highly conserved part of the heme-binding domain (Figure 1). The amino acids interacting with 2 were the following: Pro102 (strand leading to B′-helix, SRS1), Leu121 (strand running from B′-helix, SRS1), Asp301 and Ala305 (I-helix, SRS4), Val370 (K-helix, SRS5), and Leu484 (β5-sheet, SRS6). Ajmalicine (3, Figure 2). Also for the potent P450 2D6 inhibitor 3 various starting orientations were generated within the active site of the P450 2D6 protein model. Since this inhibitor of P450 2D6 is not known to be metabolized by P450 2D6, many orientations can be envisioned. The most favorable protein-inhibitor complex determined is shown in Figure 4c. In all optimized orientations the A-ring of 3 (Figure 2) was orientated above pyrrole ring B of the heme moiety. The angle between the two ring systems is ∼70°. In case 3 would be metabolized by P450 2D6, oxidation would most likely occur in the A-ring, which is supported by predictions (indicated in Figure 2) from the substrate model of P450 2D63 and which would make it a 7 Å substrate. Hydrophobic or hydrogen bonding amino acids in the protein model at a distance smaller than 5 Å from the inhibitor were part of SRS1, SRS2, SRS4 (Asp301), SRS5, and SRS6 (54) and the highly conserved part of the heme-binding domain (Figure 1). The amino acids interacting with 3 were as follows: Pro102 and Gln108 (strand leading to B′helix, SRS1), Arg115, Ser116, Leu121, and Ala122 (strand running from B′-helix, SRS1), Leu213 (F-helix, SRS2), Asp301 (I-helix, SRS4), Val370 (K-helix, SRS5), and Leu484 (β5-sheet, SRS6). GBR12909 (4, Figure 2). This relatively large substrate of P450 2D6 was recently incorporated into the substrate model of P450 2D6 (13). The observed formation of two oxidative (7 Å) metabolites by P450 2D6 could be explained with the substrate model, although, due to the large size of 4, part of the molecule extended significantly beyond the boundaries of the substrate model (8, 13). Each metabolite resulted from a different orientation (and conformation) of 4 within the substrate model (13). Both orientations of 4 could be docked into the active site of the P450 2D6 protein model in a similar relative orientation as in the substrate model. However, the orientation of 4 leading to aromatic hydroxylation led to a collision between the bis(4-fluorophenyl)methoxy moiety of 4 extending beyond the substrate model boundaries and the heme moiety of the protein model. Therefore, a conformational search was necessary to reorientate this molecular moiety prior to optimization. The orientation of 4 leading to benzylic hydroxylation could be accommodated directly in the present P450 2D6 protein model. For both metabolic reactions the site of oxidation was found to be located above pyrrole ring B of the heme moiety. The angle between the planar part of 4 and the heme moiety ranged between 70.4° and 78.7°. In both complexes (Figures 4d and 4e) the protein-substrate interactions at a distance smaller than 5 Å were found between 4 and several amino acids from SRS1, SRS2, SRS4 (Asp301), SRS5, and SRS6 (54). The orientation of 4 leading to aromatic hydroxylation also interacted with

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the highly conserved part of the heme-binding domain (Figure 1), but lacked a hydrogen bond between its basic nitrogen atom and Asp301 (Figure 4e). The amino acids interacting with both orientations of 4 were the following: Pro102 (strand leading to B′-helix, SRS1), Arg115, Ser116, Gln117, and Leu121 (strand running from B′-helix, SRS1), Leu213 (F-helix, SRS2), Asp301, Ser304, Ala305, and Thr309 (I-helix, SRS4), Val370 (K-helix, SRS5), Pro371 (β3sheet, SRS5), and Leu484 (β5-sheet, SRS6). Overall Docking Results. Some amino acids interacting with the ligands were in the core region of P450s, containing, for example, the I-helix, which is very similar in all available crystal structures (18, 21, 40, 51). Other amino acids interacting with the ligands were in regions which are poorly conserved between bacterial crystal structures (e.g., the B′-helix and the F-helix) (18, 21, 40, 51). Interactions with amino acids in the core region can be predicted with relative certainty while the predictions concerning interactions with amino acids in the poorly conserved regions remain speculative. Experimental approaches, e.g., using site-directed mutagenesis techniques, are indicated to validate the amino acids predicted to be involved in ligand binding. A Comparison between the Protein Model and the Substrate Model of P450 2D6. When superimposing substrates 1, 2, and 4 within the protein model, there seem to be two oxidation sites above the heme moiety, one above pyrrole ring A for the 5 Å substrates like 1, and one above pyrrole ring B for 7 Å substrates like 2 and 4, respectively. This suggestion is supported by docking various other substrates in the active site area of the protein model of P450 2D6 (i.e., bufuralol, codeine, sparteine (data not shown)). In the derived protein model, both described proposals for the position of the oxygen atom can be accommodated, and it is not possible, based on the available data, to identify the most likely structure for the iron-oxygen intermediate. The active site of P450 2D6 is formed by the B-, B′-, C-, F-, I-, and K-helices, the β3-, β4-, and β5-sheets, and the heme moiety. For the investigated compounds 1-4 the following amino acids were found to interact with the ligand: Pro102 and Gln108 (strand leading to B′-helix, SRS1), Arg115, Ser116, Gln117, Leu121, and Ala122 (strand running from B′-helix, SRS1), Leu213 (F-helix, SRS2), Asp301, Ser304, Ala305, and Thr309 (I-helix, SRS4), Val370 (K-helix, SRS5), Pro371 (β3-sheet, SRS5), and Leu484 (β5-sheet, SRS6). Depending on the size of the ligand, some of these interactions were absent. None of the investigated compounds interacted with SRS3, which might be due to a shift in the alignment of the G-helix (containing SRS3) relative to the alignment of Hasemann et al. (51) as described above. Asp301 is a crucial amino acid, responsible for forming an ionic hydrogen bond with a basic nitrogen atom from the substrate (or inhibitor). This has been confirmed by recent site-directed mutagenesis experiments (62), indicating the necessity of a negatively charged (acid) side chain at this position. The other amino acids predicted to interact with the substrates or inhibitor still have to be verified experimentally. The existence of two distinct sites of oxidation within the protein, together with the orientation of Asp301, would require an adjustment of the substrate model for P450 2D6 (8, 13). The substrate model incorporates only one oxidation site and two possible positions for a basic nitrogen atom of the substrates (Figure 5a) guided by the presence of two oxygen atoms within a carboxylate (Asp301) (8). The present protein model for P450 2D6,



Figure 5. Schematic representation of the relative orientation of the 5 Å (such as 1) and 7 Å substrates (such as 2 and 4, and possibly 3) (a) within the present substrate model (8) and (b) within the P450 2D6 protein model.

however, suggests the presence of two possible sites of oxidation and only one position for the basic nitrogen atom (Figure 5b). Furthermore, the orientation of Asp301 relative to the substrates docked in the active site of the protein model cannot satisfy the original proposal with two positions for the basic nitrogen atoms. In order to bring the substrate model in agreement with the protein model, the 5 Å substrates will have to be moved over a distance of approximately 2 Å relative to the 7 Å substrates. Work is in progress to examine the latter alternative more thoroughly.3

Summary A three-dimensional protein model for P450 2D6 has been constructed based on crystal structures of three bacterial P450s. The protein model is considering a wide variety of site-directed mutagenesis data concerning P450s of the P450 2 family. Three substrates and one inhibitor have been docked into the active site of the P450 2D6 protein model. The protein model suggests the possibility of two distinct oxidation sites above the heme moiety. The existence of two separate groups of substrates (either 5 Å or 7 Å) suggests that the Fe-N bridged oxygen atom proposal (with oxygen lone pairs pointing to two different oxidation sites above the heme) would be a possible model for the intermediate iron-oxygen species of P450 2D6, although the ferryl oxygen intermediate also remains a possible model, and no distinction can be made based on the data derived from this protein model. The optimized conformations and orientations of P450 2D6 ligands within the active site of the protein depend on the initial protein-ligand complex conformation. Therefore, the protein model will not be very suitable for predictive purposes regarding P450 2D6 metabolism on its own. However, it provides valuable leads for the design of chemical modification and sitedirected mutagenesis experiments. Amino acids responsible for binding and/or orientation of the various P450 2D6 substrates and inhibitors have been identified: Pro102 and Gln108 (strand leading to B′-helix, SRS1), Arg115, Ser116, Gln117, Leu121, and Ala122 (strand running from B′-helix, SRS1), Leu213 (F-helix, SRS2), Asp301, Ser304, Ala305, and Thr309 (I-helix, SRS4), Val370 (K-helix, SRS5), Pro371 (β3-sheet, SRS5), and Leu484 (β5-sheet, SRS6) and correlate well with the SRSs derived by Gotoh (54), except that none of the investigated compounds interacts with SRS3. Based on the construction of the alignment, protein-ligand interactions could be expected for Pro102, Pro103, Ser116, Gln117, Gly118, Val119, Leu121, Ala122, Leu213, Ser289, Ile297, Asp301, Ser304, Thr309, Gly367, Val370, Leu372, Val374, Met377, and Leu484 (19, 24, 62, 64, 66, 68-82, 84-90). Although not all of these amino acids were found to interact with the various ligands, several (11 out of 20) of these amino acids are seemingly involved

in protein-ligand interactions. Work is in progress to incorporate the most relevant parts of the active site area of the P450 2D6 protein model into the P450 2D6 substrate model (e.g., Asp301, the heme moiety, and the I-helix), and to further refine the orientation of the 5 Å and 7 Å substrates relative to each other.

Acknowledgment. The authors wish to thank Dr. L. M. H. Koymans for carefully reading the manuscript. Note Added in Proof. Recently a set of protein models for P450 2D6 based on a similar structural alignment and NMR derived distance restraints was published (Modi, S., Paine, M. J., Sutcliffe, M. J., Lian, L. Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1996) A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding. Biochemistry 35, 4540-4550). In both the present paper and that by Modi et al., an alignment very similar to that of Hasemann et al. (51) is used. The alignments for almost the entire structure are identical, with the exception of the exact location of some small gaps which result in very minor differences, e.g., the displacement of 1 amino acid position in certain regions. The largest differences between our alignment (shown in Figure 1) and the alignment of Modi et al. (shown in Figure 7 of Modi et al.) lie in the first part of the structure: the end of the B-helix and the beginning of the B′-helix. We aligned the strands leading to and from the B′-helix more explicitly compared to Modi et al. We used all available mutagenesis information from literature derived for other 2-family isoenzymes, while Modi et al. used NMR derived distance restraints to improve the alignment. Since both approaches give very similar results when comparing overall RMS values and amino acids interacting with the substrate(s), the work of Modi et al. is supporting the predictions based on the present homology model of P450 2D6. Supporting Information Available: Multialignment of 66 isoenzymes of the P450 2 family (Figure S1) (6 pages). Ordering information is given on any current masthead page.

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A Three-Dimensional Protein Model for Human Cytochrome P450 2D6

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