Comparison of the inward- and outward-open homology models and ligand binding of human P-glycoprotein Ilza K. Pajeva 1,2, *, Christoph Globisch 1, * and Michael Wiese 1 1 Pharmaceutical Institute, University of Bonn, Germany 2 Center of Biomedical Engineering, Bulgarian Academy of Science, Sofia, Bulgaria Introduction Since its discovery in 1976 [1], P-glycoprotein (P-gp) continues to be the main focus of research interest. In addition to its involvement in cancer multidrug resis- tance (MDR) [2], the protein acts as a protector of nor- mal tissues against xenobiotics, and is a significant factor for the absorption, distribution, metabolism and excretion of drugs [3]. These observations explain the strong research interest in P-gp, currently making it the most studied ABC transporter. A number of experimen- tal studies have been reported to date attempting to explain its structure–function relationships, broad sub- strate specificity and interactions of its ligands [4–15]. Investigations of P-gp by computational methods have developed over the years as a function of the data available for modeling. In recent years, a number of three-dimensional structures of related MDR trans- porters have become available and these data have stimulated P-gp homology modeling. Several models of the protein have been published based on the struc- tures of bacterial MDR transporters [16–19]. Keywords binding sites; homology model; ligand interactions; multidrug resistance; P-glycoprotein Correspondence M. Wiese, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany Fax: +49 228 737929 Tel: +49 228 735213 E-mail: mwiese@uni-bonn.de *These authors contributed equally to this work (Received 7 August 2009, revised 21 September 2009, accepted 29 September 2009) doi:10.1111/j.1742-4658.2009.07415.x An homology model of human P-glycoprotein, based on the X-ray struc- ture of the recently resolved mouse P-glycoprotein, is presented. The model corresponds to the inward-facing conformation competent for drug binding. From the model, the residues involved in the protein-binding cav- ity are identified and compared with those in the outward-facing confor- mation of human P-glycoprotein developed previously based on the Sav1866 structure. A detailed analysis of the interactions of the cyclic pep- tides QZ59-RRR and QZ59-SSS is presented in both the X-ray structures of mouse P-glycoprotein and the human P-glycoprotein model generated by ligand docking. The results confirm the functional role of transmem- brane domains TM4, TM6, TM10 and TM12 as entrance gates to the protein cavity, and also imply differences in their functions. The analysis of the cavities in both models suggests that the ligands remain bound to the same residues during the transition from the inward- to the outward- facing conformations. The analysis of the ligand–protein interactions in the X-ray complexes shows differences in the residues involved, as well as in the specific interactions performed by the same ligand within the same protein. This observation is supported by docking of the QZ59 ligands into human P-glycoprotein, thus aiding in the understanding of the com- plex behavior of P-glycoprotein substrates and inhibitors. The results con- firm the possibility for multispecific drug interactions of the protein, and are important for elucidating the P-glycoprotein function and ligand interactions. Abbreviations HB, hydrogen bond; MDR, multidrug resistance; MOE, Molecular Operating Environment; P-gp, P-glycoprotein; TM, transmembrane. 7016 FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS Very recently, the X-ray structure of mouse P-gp, with 87% sequence identity to human P-gp, has been refined to 3.8 A ˚ resolution [20]. The apo and drug- bound structures have been obtained that are open to the cytoplasm, thus corresponding to the inward-facing conformation of P-gp. This conformation is considered to represent the initial stage of the transport cycle competent for drug binding. The large internal cavity, formed by the bundles of the transmembrane (TM) helices, can accommodate more than one compound simultaneously, and implies a common mechanism of polyspecific drug recognition. The experimental data obtained so far on the similarities [21,22] and differ- ences [23] in substrate specificity between mouse and human P-gp, despite the strong similarity of their primary structures, raise questions regarding the way in which the co-crystallized ligands may interact with human P-gp [24]. In this article, we describe a homology model of human P-gp for the inward-facing conformation of the protein using the reported three-dimensional structure of mouse P-gp [20]. We further compare our model with the previously published homology model of P-gp [18] for the open to the extracellular space or outward- facing conformation based on the Sav1866 structure [25,26]. The comparison has been performed in rela- tion to the residues exposed to the binding cavity of the protein in both conformations. The same algorithm for the identification of the binding site residues has been applied. The analysis confirms the functional role of TM4, TM6, TM10 and TM12 for the entrance gates (portals) to the cavity, in agreement with the X-ray data, and also suggests differences in their functions. Next, the interactions of the QZ59 compounds in the X-ray structures of mouse P-gp were analyzed in detail. The analysis reveals differences in the specific interactions of each ligand with the protein. The dock- ing of the QZ59 stereoisomers into the human P-gp model, and the subsequent analysis of the ligand interactions, confirms the possibility for multispecific interactions of the ligands with the protein. Results Homology modeling of human P-gp In the template structure of mouse P-gp (PDB ID code: 3G61 chain A), the missing residues (982–1000) in the disrupted helix TM12 were replaced with the homologous part of TM6 (amino acids 339–357) by superposing the backbone atoms of the three terminal amino acids (Fig. S1). In Fig. 1, the distribution of the residues, identified as outliers in the Ramachandran plot (Fig. S2), are shown. In total, 95 outliers were identified, depicted in a space-filled rendering mode in dark yellow (Fig. 1). The homology model of the template structure was minimized using the Amber 99 force field with the ligands as environment. One hundred models were generated using the best intermediate option with medium minimization, including the prevention of clashes, to stay as close as possible to the initial structure. The final template model was selected according to the best score of the Molecular Operat- ing Environment (MOE) scoring function. A multise- quence alignment was performed by the ‘Align’ tool in MOE (see Materials and methods), including the template (PDB ID: 3G61 chain A), Swissprot data- base human MDR1 (code P08183) and closest relative to the human P-gp [hamster MDR1 (code P21448)] sequences. To obtain the final homology model, 100 structures were generated using the best intermediate option and Fig. 1. X-Ray structure of mouse P-gp (PDB ID code 3G61): distri- bution of the amino acid outliers (95 residues) identified from the Ramachandran plot (Fig. S2); the outliers are rendered in dark- yellow, space-filled mode. I. K. Pajeva et al. Comparison of open and closed models of human P-gp FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS 7017 medium minimization with the Amber 99 force field for each model to remove the bad contacts. The best model was selected according to the MOE scoring function and was investigated by the protein report function in MOE. No outliers in the TM domains of the protein were found that were important in estimat- ing the drug-binding competency. The deviating amino acids, located mostly within the loop regions of the nucleotide-binding domains (31 outliers, data not shown), were then minimized, together with the adja- cent residues, keeping the remaining protein fixed. After minimization, the outliers in the Ramachandran plot (Fig. S3) were reduced to 20. In Fig. 2, the loca- tions of the outliers within the final model of human P-gp are shown in a space-filled rendering mode in dark yellow. The rmsd of all a-carbon atoms between the homology model after minimization of the Ramachandran plot outliers and the template with the modified TM12 was 0.188 A ˚ . The protonation state of the model was assigned by the protonate 3D module in MOE, which considers the solvent accessibility and regional neighboring of the amino acids. Comparison of the cavities in the inward- and outward-facing conformations of the human P-gp models In Table 1, the residues involved in the binding cavity in the inward- and outward-facing conforma- tions of the homology model of human P-gp are shown. The residues reported for the outward-facing conformation have been identified in a previous study in which a homology model of P-gp was developed on the basis of the crystal structure of the multidrug transporter Sav1866 (see table 9 in ref. [18]). To have an equal basis for comparison, the same approach was applied for the identification of the binding pockets in the cavity of the inward- facing conformation of P-gp (the module ‘Site Finder’ in MOE, see Materials and methods). In Table 1, the residues related to the binding of the substrates dibromobimane (d), verapamil (v) and rhodamine (r) are marked, according to the experi- mental findings of Loo and coworkers [4,5,7,8,11]. Figure 3 illustrates the cavity and binding pockets identified. In Fig. 3A, the general view of the cavity and, in Fig. 3B, a closer view (from the inside) are shown. Clearly outlined are the large hydrophobic spheres occupying the entrances (portals) from the inner leaflet of the membrane to the protein cavity: the first formed by TM4 (light green) and TM6 (light magenta), and the second by TM10 (dark green) and TM12 (dark magenta). Comparing the residues reported in Table 1 for the inward- and outward-facing models, differences can be seen in the residues of the TMs exposed to the cavity in both conformations. Amino acid residues involved in the interactions with the QZ59 compounds in mouse P-gp The binding sites of the cyclic peptides QZ59 were analyzed in the X-ray structures using the ‘Ligand Interactions’ module in MOE (see Materials and meth- ods). In Table 1, for each TM, the residues involved in the interactions of the QZ59 ligands are denoted by ‘q’ in the table row ‘sub’. For the same ligand, different amino acids involved in the interactions were identified as a function of the protein molecule in the asymmetric cell unit of the crystal. For the stereoisomer QZ59-RRR (PDB ID 3G60), 11 common residues were found for both P-gp molecules (A and B) in the unit cell; mouse Tyr303 (human Tyr307), Leu335 (human L339) and Ser725 (human Ala729) were also involved in the case of molecule A (Fig. 4A), and Met68 (human Met69), Fig. 2. Homology model of human P-gp: distribution of the amino acid outliers (20 residues) identified from the Ramachandran plot (Fig. S3); outliers are rendered in dark-yellow, space-filled mode. Comparison of open and closed models of human P-gp I. K. Pajeva et al. 7018 FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS Table 1. Amino acids involved in the binding cavity of the inward (‘in’) and outward (‘out’)-facing conformations of human P-gp; the numbers and letters of the residues correspond to human P-gp; sub, residues related to substrate binding; grey color, light (in), dark (out). TM1 54: G T L A A I I H G A G L P L M M L V F G E M in a xx x x out sub b qq TM2 117: Y Y S G I G A G V L V A A Y I Q V S F W in x out sub TM3 190: I G M F F Q S M A T F F T G F I V in out sub TM4 219: L A I S P V L G L S A A V W A K I L S in out c sub v d TM5 296: N I S I G A A F L L I Y A S Y A L A F W in x x x out sub q d q TM6 330: Q V L T V F F S V L I G A F S V G Q A S P in x x x out sub q q v d q rv d q r TM7 718: A I I N G G L Q P A F A d I I F S K I I G V F in x x x x out sub q q q q TM8 762: L G I I S F I T F F L Q G F T F in x out sub q TM9 831: S R L A V I T Q N I A N L G T G I I I S F I Y in out sub r TM10 857: L T L L L L A I V P I I A I A G V V E in sub v d d TM11 938: F G I T F S F T Q A M M Y F S Y A G C F in x x x out sub v d v d q q TM12 974: V L L V F S A V d V F G A M A V G Q V S S F in x x x x x out sub q r d qq q r q r d v q d qqqq a x, residues involved in the binding pocket of the QZ59 ligands (identified by docking, see Materials and methods). b d, dibromobimane [4,5,7,8]; q, QZ59 (RRR and SSS); r, rhodamine [7,11]; v, verapamil [7,8]. c no residue involved in the outward-facing conformation. d A729 (mouse S725); V981 (mouse A977). I. K. Pajeva et al. Comparison of open and closed models of human P-gp FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS 7019 Phe71 (human Phe72) and Leu971 (human Leu975) in molecule B (Fig. 4B). Arene–arene interactions were identified with Phe332 (human Phe336) and Phe974 (human Phe978) for molecules A and B, respectively. Compared with the residues reported for QZ59-RRR in [20, fig. 3], mouse Ser725 (human Ala729) and Ala981 (human Ala985) for the ligand in molecule A, and Phe71 (human Phe72), Leu971 (human Leu975) and Ala981 (human Ala985) for the ligand in mole- cule B, were also identified, with Ala981 (human A B Fig. 3. Binding cavity in the inward-facing conformation of the homology model of human P-gp: (A) general view (face); (B) closer view (from inside). The pockets are filled with alpha spheres; grey spheres indicate hydrophobic atoms and red spheres hydrophilic atoms. The protein backbone is colored by the secondary structure; colors used for the portal TMs: light green (TM4), magenta (TM6), dark green (TM10), purple (TM12). A B Fig. 4. The interaction panel of the ligand QZ59-RRR in the X-ray structure of mouse P-gp (PDB ID code 3G60): (A) ligand 1, mole- cule A; (B) ligand 2, molecule B. The receptor exposure is shown by the size and intensity (the darker the color, the more exposed the residue) of the disks drawn behind some of the residues to denote the difference in their solvent exposure as a result of the presence of the ligand; the ligand solvent exposure is shown by smudges drawn behind some of the ligand atoms to denote the extent of solvent exposure. Symbols: , arene–arene interactions; , proximity contour; , ligand exposure; , receptor exposure. Comparison of open and closed models of human P-gp I. K. Pajeva et al. 7020 FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS Ala985) being well exposed according to the size and intensity of the disk drawn around the residue (Fig. 4A,B). Figure 5 illustrates the interactions for the QZ59-SSS stereoisomer (PDB ID 3G61). Similar to QZ59-RRR, differences have been recorded in the interactions of the ligand in the different molecules in the unit cell. For ligand 1 in molecule A, only nonspecific van der Waals’ interactions were exhib- ited (Fig. 5A), whereas, for ligand 3 in molecule B (Fig. 5C), a hydrogen bond (HB) was formed with residue Tyr303 (human Tyr307). Ligand 2 (mole- cule A) and ligand 4 (molecule B) were not fully resolved and the analyses of the ligand interactions thus involved only parts of the structures. For ligand 2 (Fig. 5B), arene–arene interactions occurred with Phe974 (human Phe978). No specific interac- tions were recorded for ligand 4 (Fig. 5D). Obvi- ously, the same ligand can occupy different positions within the protein-binding pocket. Compared with the residues reported in ref. [20, fig. 3] for QZ59-SSS, mouse Phe71, Phe728, Leu971 AC BD Fig. 5. The interaction panel of the ligand QZ59-SSS in the X-ray structure of mouse P-gp (PDB ID code 3G61): (A) ligand 1, molecule A; (B) ligand 2, molecule A; (C) ligand 3, molecule B; (D) ligand 4, molecule B. The receptor exposure is shown by the size and intensity (the darker the color, the more exposed the residue) of the disks drawn behind some of the residues to denote the difference in their solvent exposure as a result of the presence of the ligand; the ligand solvent exposure is shown by smudges drawn behind some of the ligand atoms to denote the extent of solvent exposure. Symbols: , arene–arene interactions; , proximity contour; , ligand exposure; , receptor exposure. I. K. Pajeva et al. Comparison of open and closed models of human P-gp FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS 7021 and Ile977 (Fig. 5B) were also identified, with Tyr303 performing a specific HB interaction (Fig. 5C). Amino acid residues involved in the interactions with QZ59 compounds in the homology model of human P-gp The analysis of the residues involved in the interac- tions was performed in two ways: (a) using the QZ59- SSS ligands as incorporated from the X-ray sources; and (b) using docking (by GOLD, see Materials and methods) to better explore the binding possibilities of the ligands. In the homology model of human P-gp, the same amino acids as in the X-ray structures inter- acted with the QZ59-SSS ligand, and two additional residues were identified: human Gln725 and Met986 (data not shown). The docking of the QZ59 stereoisomers yielded sta- ble and similar solutions. Several runs were performed using either one or two ligands simultaneously to define the binding pocket. The poses were fully repro- ducible with close GoldScore values. The top score poses were used for further analysis. In Table 1, the residues involved in the binding sites of the QZ59 ligands identified in the best docking poses in the human P-gp inward-open model are marked by ‘x’. Figure 6 visualizes the interaction of QZ59-RRR and QZ59-SSS with the residues in the human P-gp model. Figure 6A shows the general view of the best docked poses of both stereoisomers in the two binding sites superimposed on the P-gp backbone. Similar to the SSS enantiomer, the RRR enantiomer could poten- tially occupy the two binding sites. Figure 6B shows a closer view of the interactions of the two peptides in the lower located binding site, and Fig. 6C in the upper site. Comparing the residues comprising the binding sites of the ligands in human and mouse P-gp (Table 1: ‘q’ with ‘x’; Figs 4 and 5 with Fig. 6), simi- larities and differences in relation to the particular amino acids involved and their specific interactions can be outlined. The residues in the ligand-binding sites in Fig. 6. (A) A front overview of the P-gp binding cavity with the inhibitors QZ59-RRR and QZ59-SSS (in space-filling form) docked into the two binding sites and superimposed on the P-gp backbone (grey line). (B) Closer view (from the bottom) of the protein–ligand interactions and the surface of the binding site in the lower binding site. (C) Closer view (from the bottom) of the protein–ligand interac- tions and the surface of the binding site in the upper binding site. The Gaussian contact surface is presented with the following color coding: green, hydrophobic; magenta, HB; blue, mildly polar. The HB distance and score are colored in magenta; the residues are col- ored in green; the structures of the ligands are rendered in stick form and colored according to the atom types; the HB atoms are shown as balls and the distance between (=O) of QZ59-RRR and (–N<) of Gln725 is 2.84 A ˚ (64% score, see Materials and methods). A B C Comparison of open and closed models of human P-gp I. K. Pajeva et al. 7022 FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS the X-ray structures of mouse P-gp partially overlap with those in the docked poses in the human P-gp model. Although Tyr303 (human Tyr307) performs an HB interaction with QZ59-SSS in the X-ray structure (Fig. 5C), human Gln725 is involved in an HB–donor interaction in human P-gp, and Phe343 can perform arene–arene interactions with QZ59-RRR (Fig. 6B). Thus, it can be proposed that the QZ59 ligands will bind to human P-gp in a similar, but not identical, manner as to mouse P-gp, interacting specifically with different residues from the same environment. Such a result is not surprising considering the observed differ- ences mentioned above in the interactions of the same ligand with two different molecules of the same protein in the X-ray structures (Figs 4 and 5). Notably, the hydrophobic residues with the human P-gp codes Phe336, Phe343, Phe728, Phe978 and Val982 are the most involved amino acids in both the X-ray and docked poses of the ligands. The same residues have been proven experimentally to relate to other P-gp substrates [4,7,8,10,11,14]. Discussion For the different TMs, differences between the residues exposed to the cavity in both the open and closed con- formations have been observed. The most striking dif- ference is the absence of amino acids of TM4 and TM10 facing the cavity. Although residues of TM4 and TM10 are broadly accessible in the inward-open conformation, they are fully buried in the outward- open form (Table 1). At the same time, the same residues of TM6 and TM12 face the cavity in both conformations and, in addition, these domains possess the highest number of experimentally proven amino acids involved in drug binding. Among them are a number of hydrophobic residues, such as human Phe336, Phe339, Phe340, Phe343, Phe978, Val981, Val982, Ala985, identified in the binding sites of the QZ59 ligands; moreover, some have been found to per- form specific (arene–arene) interactions, such as Phe336 (mouse Phe332, Fig. 4A), Phe978 (mouse Phe974, Figs 4B and 5B) and Phe343 (Fig. 6B). TM6 and TM12 are also the most involved domains in interactions with other P-gp substrates, such as verapa- mil, rhodamine and dibromobimane, proven by drug-binding experiments (Table 1, row ‘sub’). In the most recent study, Loo et al. [5] also found the largest number of mutations in TM6 and TM12 in arginine- scanning mutagenesis experiments. Considering that these TMs form the two portals (TM4–TM6 and TM10–TM12) for entering the cavity from the inner leaflet of the membrane, the differences observed above also suggest differences in their role for the function of protein and ligand binding. TM4 and TM10, being parts of the portals, could be involved in weaker interactions with the entering ligands that are lost during the transport cycle. In Table 1, three resi- dues only are reported as belonging to these domains, related to interactions with P-gp substrates: human Ser222 (TM4), Ile868 and Gly872 (TM10) (Table 1). Figuratively, TM4 and TM10 could function as ‘portal keepers’, preventing the substances that enter the cav- ity from the inner leaflet of the membrane escaping back. In contrast, TM6 and TM12 could be regarded as the ‘portal carriers’, being mainly responsible for ligand interactions. Interestingly, the TM6 and T12 residues mostly perform hydrophobic interactions, whereas the more specific HB-type interactions could be related to other domains, such as, for example, Tyr307 (mouse Tyr303) of TM5 (Fig. 5C) and Gln725 of TM7 (Fig. 6A). Studying the interactions of specific P-gp inhibitors, representatives of the third-generation MDR modulators, we found specific HB interactions with Tyr117 (TM2), Tyr307 (TM5) and an arene– arene-type interaction with Tyr953 (TM11) [27], thus outlining, in addition, the role of TM2, TM5, TM7 and TM11 in ligand interactions. It is worth noting that these residues also face the cavity in both confor- mations of the protein (Table 1). From the above analysis, it is most likely that the ligands remain bound to the same residues during the transition from the inward- to the outward-facing con- formation of the protein, suggesting that the ligand is not flipped. Next, the detailed analyses of the ligand interactions with the protein, as recorded here for the QZ59 com- pounds (Figs 4 and 5), show differences in the residues involved, as well as in the specific interactions of the same ligand with the same protein. For QZ59-RRR, Phe332 (human Phe336) is in a favorable position for arene–arene interactions in molecule A, whereas Phe974 (human Phe978) is involved in such interac- tions in molecule B (Fig. 4A,B); Tyr303 (human Tyr307) forms an HB interaction with ligand 3 in mol- ecule B (Fig. 5C); at the same time, no such interac- tion is recorded for ligand 1 in molecule A (Fig. 5A). Whether these differences can be related to the experi- mental conditions under which the ligands were co-crystallized, or reflect the possibility for different binding locations and orientations of the same ligand in the same protein environment, remains to be proven. The docking of QZ59 ligands into the human P-gp binding cavity supports the latter suggestion. The results confirm the possibility for binding of the same ligand in two different binding sites, as shown for I. K. Pajeva et al. Comparison of open and closed models of human P-gp FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS 7023 QZ59-RRR in Fig. 6B,C. This observation, illustrated here by the analysis of the X-ray complexes and sup- ported by binding simulation, could help, for example, to better understand the complex behavior of P-gp substrates and inhibitors in functional assays. In conclusion, the results of this study confirm the possibility for multispecific interactions of the protein with its ligands, and aid in the elucidation of P-gp function and drug interactions. Materials and methods Homology modeling Chain A from the crystal structure of mouse P-gp (PDB ID: 3G61 [20]) was used as template for the homology model. The Swissprot database sequences of human P-gp (P08183) and hamster P-gp (P21448) were used for alignment [28]. The alignment was obtained with the ‘Align’ tool in MOE [29], using the default tree-based approach with the blosum62 substitution matrix and increased values for the gap penalty of 15 and gap extension penalty of 2. The homology model was calculated by the ‘Homology Model’ method in MOE using a rotamer library and loop dictionary derived from the X-ray structures to predict the coordinates of deviating residues. The best intermediate approach with the medium minimization option and the Amber 99 force field was used to derive the model. The co- crystallized ligand structures were defined as environment and kept fixed. The best model (out of 100) was selected according to the GB ⁄ VI scoring function (Coulomb and generalized Born interaction energies of the model and environment). The stereochemical quality of the model was inspected by the protein report of MOE. The MOE-ProSu- perpose module was used to calculate the rmsd values. Identification of the binding pockets The ‘Site Finder’ tool in MOE [29] was employed for the identification of the binding sites in the inward-open con- formation of the P-gp homology model. The program is based on the methodology of convex hulls which produces pockets invariant to rotation of the atomic coordinates. It treats the set of three-dimensional points by triangulation and associates each resulting simplex with a sphere, coded as ‘alpha sphere’. The radius of the sphere is proportional to the convex hull of the point set. Each sphere is classified as either ‘hydrophobic’ or ‘hydrophilic’ depending on whether the sphere is a good HB point in the receptor. Hydrophilic spheres not close to hydrophobic ones are eliminated as they generally correspond to water sites. The generated pockets consist of one or more alpha spheres, and at least one is hydrophobic. The following settings were used: radius of a hydrophilic HB sphere, 1.4 A ˚ ; radius of a hydrophobic sphere, 1.8 A ˚ ; isolate donor–acceptor distance, 3 A ˚ ; connection distance between clusters of alpha spheres, 2.5 A ˚ ; minimum site size, 3; minimum site radius, 2 A ˚ . Docking of the QZ59 ligands into human P-gp models The QZ59 ligands were prepared in Sybyl [30]. QZ59-RRR was extracted from its complex 3G60 and QZ59-SSS from 3G61. The chirality and atom types of the compounds were checked; the missing hydrogen atoms were added, and the geometries were optimized with the Tripos force field and Gasteiger–Hueckel charges. The minimized structures were subsequently exported as mol2 files for docking with the GOLD Suite [31,32], which applied a genetic optimization algorithm for docking flexible ligands into protein-binding sites. The binding pocket was defined on the basis of the co-crystallized ligands in the X-ray structure. Considering the substantial volume of the binding cavity (around 6000 A ˚ 3 [20]), the pocket was extended by 10 A ˚ and out- ward-facing amino acids were deselected. The default setting for the genetic algorithm and the original GoldScore function were used to rank the ligand poses. Docking was performed by the ‘slow (most accurate)’ option for balance between speed and accuracy. Analysis of the ligand interactions The X-ray complexes (mouse P-gp) and docked poses (human P-gp) of the QZ59 compounds were analyzed by the ‘Ligand Interactions’ tool in MOE. The method imple- mented is fully described in [33]. The information content displayed in the ligand interactions panel consists of the selected ligand and the receptor-interacting entities, namely HB residues, close but non-bonded residues (approaching the ligand but not having any strong interactions, i.e. HBs), solvent molecules and ions. The solvent-accessible surface area and the ligand proximity outline were also estimated. HBs were assigned to each pair of heavy atoms from the ligand and receptor according to probability criteria derived from a large training set [34,35]. The HB scores were expressed as percentages and the HB directionality was noted. The ligand and residue solvent accessibility metrics were estimated by measuring the exposed surface area once each of the atoms had been assigned a van der Waals’-like radius of +1.4 A ˚ (water solvent). The solvent exposure of receptor residues was calculated by examining the difference between the solvent-exposed surface area of the receptor with and without the presence of the ligand. For the ligands, the surface accessibility calculation was carried out on the ligand + receptor complex. The default settings were applied for the definition of hydrogen-bonded and proximity interactions. Comparison of open and closed models of human P-gp I. K. Pajeva et al. 7024 FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS Acknowledgements I.P. and M.W. gratefully acknowledge the generous support by the Alexander von Humboldt Foundation, Germany. I.P also thanks the National Science Foun- dation of Bulgaria. References 1 Juliano RL & Ling V (1976) A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455, 152–162. 2 Sharom FJ (2008) ABC multidrug transporters: struc- ture, function and role in chemoresistance. Pharmacoge- nomics 9, 105–127. 3 Glavinas H, Krajcsi P, Cserepes J & Sarkadi B (2004) The role of ABC transporters in drug resistance, metab- olism and toxicity. Curr Drug Deliv 1, 27–42. 4 Loo TW & Clarke DM (1997) Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate. 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J Med Chem 52, 3328–3341. 23 Tang-Wai DF, Kajiji S, DiCapua F, de Graaf D, Roninson IB & Gros P (1995) Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distin- guished by their respective drug resistance profiles and sensitivity to modulators. Biochemistry 34, 32–39. 24 Gottesman MM, Ambudkar SV & Xia D (2009) Structure of a multidrug transporter. Nat Biotechnol 27, 546–547. 25 Dawson RJ & Locher KP (2006) Structure of a bacte- rial multidrug ABC transporter. Nature 443, 180–185. 26 Dawson RJ & Locher KP (2007) Structure of the multi- drug ABC transporter Sav1866 from Staphylococcus I. K. Pajeva et al. Comparison of open and closed models of human P-gp FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS 7025 [...]... Backbone of the TMs in the X-ray structure of mouse P-gp (PDB ID code 3G61) with the disrupted helix in TM12 and the superposed part of TM6 (blue, on the left of the cavity) used as a template for the modified TM12 Fig S2 Ramachandran plot of the X-ray structure of chain A (PDB ID code 3G61) Fig S3 Ramachandran plot of the homology model of human P-gp This supplementary material can be found in the online.. .Comparison of open and closed models of human P-gp 27 28 29 30 31 32 33 34 I K Pajeva et al aureus in complex with AMP-PNP FEBS Lett 581, 935–938 Pajeva IK, Globisch C & Wiese M (2009) Combined pharmacophore, docking and 3D QSAR study of inhibitors of ABCB1 and ABCC1 transporters ChemMedChem 4, 1883– 1896 Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD & Bairoch A (2003) ExPASy: the proteomics... the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal... P (2007) 2D depiction of protein ligand complexes J Chem Inf Model 47, 1933–1944 Labute P, Williams C, Feher M, Sourial E & Schmidt JM (2001) Flexible alignment of small molecules J Med Chem 44, 1483–1490 7026 35 Labute P (2001) Probabilistic receptor potentials J Chem Comput Group, http://www.chemcomp.com/journal/cstat htm [accessed on 12 October 2009] Supporting information The following supplementary... server for in-depth protein knowledge and analysis Nucleic Acids Res 31, 3784–3788 MOE 2008.10 (Molecular Operating Environment) Chemical Computing Group, Montreal, Quebec SYBYL 8.1 Tripos Inc., St Louis, MO GOLD Version 4.0.1 The Cambridge Crystallographic Data Centre, Cambridge, UK Jones G, Willett P, Glen RC, Leach AR & Taylor R (1997) Development and validation of a genetic algorithm for flexible docking... delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 7016–7026 ª 2009 The Authors Journal compilation ª 2009 FEBS . protein ligand interactions and the surface of the binding site in the lower binding site. (C) Closer view (from the bottom) of the protein ligand interac- tions and the surface of the binding site in the upper binding. some of the residues to denote the difference in their solvent exposure as a result of the presence of the ligand; the ligand solvent exposure is shown by smudges drawn behind some of the ligand. Comparison of the inward- and outward-open homology models and ligand binding of human P-glycoprotein Ilza K. Pajeva 1,2, *, Christoph Globisch 1, * and Michael Wiese 1 1