Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

  • Aina W Ravna1Email author,

    Affiliated with

    • Ingebrigt Sylte1 and

      Affiliated with

      • Georg Sager1

        Affiliated with

        Theoretical Biology and Medical Modelling20096:20

        DOI: 10.1186/1742-4682-6-20

        Received: 4 June 2009

        Accepted: 4 September 2009

        Published: 4 September 2009

        Abstract

        The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.

        Introduction

        The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 belong to the ABC superfamily, a subgroup of Primary active transporters [1]. The transporters in the ABC superfamily are structurally related membrane proteins that have a common intracellular motif that exhibits ATPase activity. This motif cleaves ATP's terminal phosphate to energize the transport of molecules from regions of low concentration to regions of high concentration [13]. Since ABC genes are highly conserved between species, it is likely that most of these genes have been present since the beginning of eukaryotic evolution [4].

        The overall topology of ABCB1, ABCC4 and ABCC5 is divided into transmembrane domain 1 (TMD1) - nucleotide-binding domain 1 (NBD1) - TMD2 - NBD2 (Figure 1). The Walker A, or phosphate binding loop (P-loop), and Walker B motifs, are localized in the NBDs, while the TMDs contribute to the substrate translocation events (recognition, translocation and release). ABCB1, ABCC4 and ABCC5 are exporters, pumping substrates out of the cell.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig1_HTML.jpg
        Figure 1

        Overall domain topology of ABCB1, ABCC4 and ABCC5.

        Transporters have drug recognition sites that make them specific for particular substrates, and drugs may interact with these recognition sites and either inhibit the transporter or act as substrates. Experimental studies have shown that ABCB1 transports cationic amphiphilic and lipophilic substrates [58], while ABCC4 and ABCC5 transport organic anions [9]. Both ABCC4 and ABCC5 transport cAMP and cGMP, however, with differences in their kinetic parameters; ABCC4 with a preference for cAMP and ABCC5 with a preference for cGMP [9, 10].

        When chemotherapeutic agents are expelled from cancer cells as substrates of ABCB1, ABCC4 or ABCC5, the result is multidrug resistance. In order to overcome multidrug resistance, development of inhibitors of drug efflux transporters has been sought for use as supplement to drug therapy [11]. However, clinical trials of potential anti-MDR agents have been disappointing due to adverse effects in vivo of agents being very effective in vitro. Even if there is a long time since Victor Ling described MDR, (i.e. ABCB1) [12], very little is known about subtype selective recognition and binding of ABC proteins. Structural insight into their mode of ligand interaction and functional mechanisms will be an important contribution to pinpoint potential drug targets and to design putative inhibitors. Recent papers report a considerable difference in substrate specificity of ABCC4 and ABCC5 [9], including various chemotherapeutic agents [13], and with potential impact on reversal of MDR [14]. Elucidating the molecular aspects of ligand interactions with ABCB1, ABCC4 or ABCC5 may therefore aid in the design of therapeutic agents that can help to overcome multidrug resistance.

        We have previously constructed molecular models of ABCB1 [15], ABCC4 [16] and ABCC5 [17] based on the Staphylococcus aureus ABC transporter Sav1866, which has been crystallized in an outward-facing ATP-bound state [18]. In this study, we present molecular models of ABCB1, ABCC4 and ABCC5 based on a wide open inward-facing conformation of Escherichia coli MsbA [19]. Since the molecular modelling was carried out before the X-ray crystal structure of the Mus musculus ABCB1 in a drug-bound conformation was published [20], we got a unique opportunity to test our methodology, molecular modelling by homology, and the quality of the ABCB1 model, when the crystal structure was published. Since we wanted to elucidate differences in the electrostatic and molecular features of the drug recognition conformation of these transporters, the wide open conformation of the MsbA template [19] was of particular interest. The electrostatic potential surfaces (EPS) of the models were calculated, and the models were compared to the X-ray crystal structure of the Mus musculus ABCB1 [20], and with published cross-linking and site directed mutagenesis data on ABCB1 [2135].

        Computational methods

        Software

        Version 3.4-9b of the Internal Coordinate Mechanics (ICM) program [36] was used for homology modelling, model refinements and electrostatic calculations. The AMBER program package version 8.0 [37] was used for molecular mechanics energy minimization.

        Alignment

        A multiple sequence alignment of (SWISS-PROT accession numbers are given in brackets) human ABCB1 (P08183), human ABCC4 (O15439), human ABCC5 (O15440), human ABCC11 (Q9BX80), Escherichia coli MsbA (P60752) and Vibrio cholerae MsbA (Q9KQW9), obtained using T-COFFEE [38], Version 4.71 available at the Le Centre national de la recherche scientifique website http://​www.​igs.​cnrs-mrs.​fr/​Tcoffee/​tcoffee_​cgi/​index.​cgi, was used as a basis for the homology modelling module of ICM program [36]. ABCC11 was included in the alignment because it is closely related to ABCC5 phylogenetically [15], and its inclusion may strengthen the alignment. The alignment was adjusted for sporadic gaps in the TMH segments, and for secondary structure predictions defining the boundaries of the TMHs using the PredictProtein server for sequence analysis and structure prediction [39], and SWISS-PROT [40].

        The alignment of human ABCB1 and Escherichia coli MsbA was compared to previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], and it was observed that in our alignment, the ABCB1 sequence was shifted 2 positions to the left relative to the E. coli MsbA sequence in the alignment of TMH2, and 1 position the left relative to the E. coli MsbA sequence in the alignment of TMH6, as compared to the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41]. Thus, 3 alignments were used to construct 3 ABCB1 models, 1 model with our original alignment, 1 model with TMH2 adjusted to correspond to the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], and 1 model with both TMH2 and TMH6 adjusted, thus using the same alignment as the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41]. The alignment of Escherichia coli MsbA, human ABCB1, human ABCC4 and human ABCC5 used for the homology modelling procedure, with TMH2 adjusted to correspond to the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], is shown in Figure 2. For illustrative purposes, only the sequences of the template and the 3 target proteins ABCB1, ABCC4 and ABCC5 are shown.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig2_HTML.jpg
        Figure 2

        Alignment of Escherichia coli MsbA, human ABCB1, human ABCC4 and human ABCC5 used as input alignment for the ICM homology modelling module. TMHs, Walker A motifs and Walker B motifs are indicated as boxes.

        Homology modelling

        A full atom version of the open inward facing Escherichia coli MsbA X-ray crystal structure (PDB code: 3B5W[19]) was kindly provided by Geoffery Chang and used as a template in the construction of the homology models of ABCB1, ABCC4 and ABCC5. The ICM program constructs the molecular model by homology from core sections defined by the average of Cα atom positions in conserved regions. Loops were searched for within several thousand structures in the PDB databank [42] and matched in regard to sequence similarity and sterical interactions with the surroundings of the model, and the best-fitting loop was selected based on calculating the maps around the loops and scoring of their relative energies. The segment connecting NBD1 and TMD2 was also included in the loop search procedure.

        Calculations

        The ABCB1, ABCC4 and ABCC5 models were refined by globally optimizing side-chain positions and annealing of the backbone using the RefineModel macro of ICM. The macro was comprised of (1) a side-chain conformational sampling using 'Montecarlo fast' [43], (2) 5 iterative annealings of the backbone with tethers (harmonic restraints pulling an atom in the model to a static point in space represented by a corresponding atom in the template), and (3) a second side-chain conformational sampling using 'Montecarlo fast'. 'Montecarlo fast' samples conformational space of a molecule with the ICM global optimization procedure, and its iterations consist of a random move followed by a local energy minimization, and calculation of the complete energy. The iteration is accepted or rejected based on energy and temperature.

        The refined ABCB1, ABCC4 and ABCC5 models were energy minimized using the AMBER 8.0 program package [37]. Two energy minimizations were performed for each model, (1) with restrained backbone by 500 cycles of the steepest descent minimization followed by 500 steps of conjugate gradient minimization, and (2) with no restraints by 1000 cycles of the steepest descent minimization followed by 1500 steps of conjugate gradient minimization. The leaprc.ff03 force field [37], and a 10 Å cut-off radius for non-bonded interactions and a dielectric multiplicative constant of 1.0 for the electrostatic interactions, were used in the molecular mechanics calculations.

        The EPS of the ABCB1, ABCC4 and ABCC5 models were calculated with the ICM program, with a potential scale from -10 to +10 kcal/mol.

        Model validation

        To check the stereochemical qualities of the ABCB1, ABCC4 and ABCC5 models, the SAVES Metaserver for analyzing and validating protein structures http://​nihserver.​mbi.​ucla.​edu/​SAVES/​ was used. Programs run were Procheck [44], What_check [45], and Errat [46], and the pdb file of the open inward facing Escherichia coli MsbA template [19] was also checked for comparison with the models.

        For further validation, the ABCB1, ABCC4 and ABCC5 models were compared with the X-ray crystal structure of the Mus musculus ABCB1 [20] and cross-linking and site directed mutagenesis data published on ABCB1 [2135].

        Results

        The 3 ABCB1 models, constructed based on 3 different alignments, where compared with cross-linking data and subsequently also the X-ray crystal structure of the Mus musculus ABCB1 [20], and it was revealed that when TMH2 was aligned as the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], amino acids in TMH2/TMH11 (Val133/Gly939 and Cys127/Ala935) where oriented towards each other in accordance with both cross-linking data and the X-ray crystal structure of the Mus musculus ABCB1 [20]. However, when TMH6 was aligned as the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], ligand binding amino acids (Ile340 and Phe343) pointed away from the drug binding site, while when aligned as proposed from our T-COFFEE [38] alignment, it was in accordance both with cross-linking data and the X-ray crystal structure of the Mus musculus ABCB1 [20]. Thus, the ABCB1 model which was most in accordance with cross-linking data and the X-ray crystal structure of the Mus musculus ABCB1 [20] was based on the alignment where TMH2 was adjusted according to the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], while TMH6 was kept exactly as in our T-COFFEE [38] alignment. The alignment of Escherichia coli MsbA, human ABCB1 (TMH2 adjusted), human ABCC4 and human ABCC5 used for the homology modelling procedure is shown in Figure 2. For illustrative purposes, only the sequences of the template and the 3 target proteins ABCB1, ABCC4 and ABCC5 are shown.

        The energy minimized ABCB1, ABCC4 and ABCC5 models are shown in Figures 3A-C. Each transporter was in an open V-shaped inward conformation with their NBD1 and NBD2 ~50 Å apart. Both Walker A motifs of each model consisted of a coiled loop and a short α-helix (P-loop), and the ATP-binding half sites faced each other. The Walker B motifs were in β-sheet conformation and localized in the NBD's hydrophobic cores, which were constituted of 5 parallel β-sheets. The amino acids localized on the surface of each NBD were mainly charged. In the "arms" of the V-shaped structure, NBD1 was associated with TMHs 1, 2, 3 and 6 (TMD1), and TMHs 10 and 11 (TMD2), while NBD2 was associated with TMHs 4 and 5 (TMD1), and TMHs 7, 8, 9 and 12 (TMD2). Thus, the TMDs were twisted relative to the NBDs, such that TMH4 and TMH5 were crossed over ("cross-over motif" [19]) and associated with TMD2, and TMH10 and TMH11 were crossed over and associated with TMD1. All TMHs contributed to substrate translocation pore, which was closed towards the extracellular side.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig3_HTML.jpg
        Figure 3

        Backbone Cα-traces of ABCB1 model (A), ABCC4 model (B) and ABCC5 model (C) viewed in the membrane plane, cytoplasm downwards. Colour coding: blue via white to red from N-terminal to C-terminal.

        The loop connecting NBD1 and TMD2 of each transporter was abundant with charged amino acids. The loop connecting NBD1 and TMD2 of ABCB1 was in extended conformation forming a β-sheet between amino acids sections Lys645-Glu652 and Lys665-Ser671, while the loops connecting the subunits of ABCC4 and ABCC5 were α-helical. ABCB5 featured an insertion loop (as compared with the amino acid sequences of Escherichia coli MsbA) from Ile479 to His548 in NBD1, and as displayed in Figures 3C and 4C, this loop was pointing away from NBD1 parallel to the membrane. However, modelling loops of lengths as that of the connection between NBD1 and TMD2 is relatively inaccurate and consequently the modelled loop structures must be regarded as uncertain.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig4_HTML.jpg
        Figure 4

        A-C : Backbone Cα-traces of ABCB1 model (A), ABCC4 model (B) and ABCC5 model (C) viewed from intracellular side. Colour coding: blue via white to red from N-terminal to C-terminal. D - F: The water-accessible surfaces of ABCB1 model (D), ABCC4 model (E) and ABCC5 model (F) viewed from intracellular side collared coded according to the electrostatic potentials 1.4 Å outside the surface; negative (-10 kcal/mol), red to positive (+10 kcal/mol), blue. G-I: Cross sections along the inner membrane layer of water-accessible surfaces of ABCB1 model (G), ABCC4 model (H) and ABCC5 model (I) viewed from intracellular side, colour coding as D-F. All illustrations are in similar view.

        Figures 4A-I show the EPS of the substrate recognition area of each of the ABC models. The EPS of the substrate recognition area in the TMDs of ABCB1 was neutral with negative and weakly positive areas, while the EPS of the ABCC5 substrate recognition area was generally positive. The substrate recognition area of ABCC4 was generally positive with negative area "spots".

        The results from the stereochemical validations retrieved from the SAVES Metaserver http://​nihserver.​mbi.​ucla.​edu/​SAVES/​ are shown in Table 1. Overall factors from the Errat option at ~90 indicate that the models were of high quality.
        Table 1

        Results from the stereochemical validations retrieved from the SAVES Metaserver

         

        Errat

        Procheck (%)

        Whatcheck

          

        Core

        Allow

        Gener

        Disall

         

        ABCB1

        89.7

        80.2

        15.1

        3.4

        1.3

        Satisfactory

        ABCC4

        86.7

        81.1

        14.4

        3.0

        1.5

        Satisfactory

        ABCC5

        90.6

        80.5

        15.1

        2.7

        1.7

        Satisfactory

        Escherichia coli MsbA[19]

        58.2

        54.9

        37.4

        5.6

        2.1

        Satisfactory

        Site directed mutagenesis studies on ABCB1 have indicated that Ile306 (TMH5) [27, 35], Ile340 (TMH6) [33], Phe343 (TMH6) [21, 27], Phe728 (TMH7) [27], and Val982 (TMH12) [33, 35] may participate in ligand binding. As shown in Figure 5A, these residues may form a substrate recognition site in the ABCB1 model. The involvement of these residues in ligand binding is confirmed in the X-ray crystal structure of the Mus musculus ABCB1 [20] (Figure 5B). Table 2 shows the corresponding residues in ABCC4 and ABCC5. Measured Cα-Cα distances in the human ABCB1 model, in the X-ray crystal structure of the Mus musculus ABCB1 [20] and experimental distance ranges from cross-linking studies and are listed in Table 3.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig5_HTML.jpg
        Figure 5

        Comparison of proposed drug binding site in ABCB1 model (A) and the drug binding site in the X-ray crystal structure of P-glycoprotein (ABCB1) [20] (B) viewed from the intracellular side with amino acids suggested from site directed mutagenesis studies to take part in ligand binding displayed as sticks coloured according to atom type (C = grey; H = dark grey; O = red; N = blue); Ile306 (TMH5) [27, 35], Ile340 (TMH6) [33], Phe343 (TMH6) [21, 27], Phe728 (TMH7) [27], and Val982 (TMH12) [33, 35]. Amino acids in panel B are numbered according to human ABCB1. Mus musculus numbering: Ile302, Ile336, Phe339 Phe724 and Val978. Differences in helix tilting in the panels refer to the different conformations of ABCB1, outward facing conformation in the left panel and closed conformation in the right panel.

        Table 2

        Human ABCB1 amino acid residues shown to interact with ligands in site directed mutagenesis studies, corresponding Mus musculus ABCB1 amino acids shown to interact with ligand in X-ray crystal structure [20], and corresponding amino acid residues in ABCC4 and ABCC5.

        TMH

        Human ABCB1

        Mus musculus ABCB1[20]

        ABCC4

        ABCC5

        1

        Leu65 [26]

        Leu64

        Glu103

        Gln190

        5

        Ile306 [27, 35]

        Ile302*

        Ser328

        Val410

        6

        Ile340 [33]

        Ile336

        Gly359

        Asn441

        6

        Phe343 [21, 27]

        Phe339

        Arg362

        Thr444

        7

        Phe728 [27]

        Phe724

        Ala727

        Ser872

        12

        Val982 [33, 35]

        Val978

        Leu987

        Val1137

        *) Not direct contact with ligand in Mus musculus ABCB1 X-ray crystal structure [20].

        Table 3

        Comparison of Cα-Cα distances in the human ABCB1 model, Cα-Cα distances in the Mus musculus ABCB1 X-ray crystal structure [20] and distances between residues from experimental cross-linking studies on ABCB1.

        Region

        Residues Human ABCB1 (Mus musculus ABCB1)

        Cα-Cα distances (Å)

        Exp. Cross-linking (Å)

        Ref

          

        Human ABCB1 model

        Mus musculus ABCB1 (Pdb code: 3G60)

          

        TMH1/TMH11

        M68/Y950 (M67/Y946)

        12.9

        9

         

        [25]

         

        M68/Y953 (M67/Y949)

        15.6

        10

          
         

        M68/A954 (M67/A950)

        17.3

        11.2

          
         

        M69/A954 (M68/A950)

        19

        11.2

          
         

        M69/F957 (M68/F953)

        19.5

        13

          

        TMH2/TMH11

        V133/G939 (V129/G935)

        6.2

        5

         

        [24]

         

        C137/A935 (C133/A931)

        7.4

        5.1

          

        TMH4/TMH10

        S222/I868 (S218/I864)

        34.7

        30.3

        9-25

        [34]

         

        S222/G872 (S218/G868)

        35

        30.8

          

        TMH4/TMH12

        L227/S993 (L223/S989)

        32.3

        22.8

        5.5-15

        [30]

         

        V231/S993 (I227/S989)

        31.2

        20.8

          
         

        W232/S993 (W228/S989)

        28.9

        20.1

          
         

        A233/S993 (A229/S989)

        26

        16.3

          
         

        I235/S993 (I231/S989)

        30

        19.9

          
         

        L236/S993 (L232/S989)

        26.6

        18

          

        TMH5/TMH8

        N296/G774 (N292/G770)

        6.9

        8.1

         

        [23]

         

        I299/F770 (M295/F766)

        8.9

        9.3

          
         

        I299/G774 (M295/G770)

        11.9

        10.7

          
         

        G300/F770 (G296/F766)

        7.2

        7.7

          

        TMH5/TMH10

        I306/I868 (I302/I864)

        33

        29.9

        13-25

        [34]

         

        I306/G872 (I302/G868)

        34.3

        29.6

          

        TMH5/TMH11

        I306/T945 (I302/T941)

        33.4

        31.3

        13-25

        [34]

        TMH5/TMH12

        I306/V982 (I302/V978)

        19

        19.8

        13-25

        [34]

         

        I306/A985 (I302/A981)

        22.4

        20.5

          

        TMH5/TMH12

        A295/S993 (A291/S989)

        22.7

        16.3

        5.5-15

        [30]

         

        I299/S993 (M295/S989)

        21.4

        13.7

          

        TMH6/TMH7

        L339/F728 (L335/F724)

        18.8

        16.3

        20-25

        [28]

        TMH6/TMH10

        P350/V874 (P346/V870)

        34.1

        23.2

        5.5-15

        [30]

         

        P350/E875 (P346/E871)

        31.6

        20.1

          
         

        P350/M876 (P346/M872)

        28.9

        18.3

          

        TMH6/TMH10

        L339/I868 (L335/I864)

        27

        23.4

        13-25

        [34]

         

        L339/G872 (L335/G868)

        27.9

        24

          
         

        L332/Q856 (L328/Q852)

        31.9

        24.9

          

        TMH6/TMH11

        P350/G939 (P346/G935)

        23.5

        20.9

        5.5-15

        [30]

        TMH6/TMH11

        L339/T945 (L335/T941)

        25

        22.7

        20-25

        [32]

        TMH6/TMH11

        L339/F942 (L335/F938)

        24.9

        23.9

        25

        [34]

        TMH6/TMH12

        L332/L975 (L328/L971)

        10.7

        12.5

        5.5-15

        [28]

        TMH6/TMH12

        F343/M986 (F339/M982)

        20.4

        15.2

         

        [29]

         

        G346/G989 (G342/G985)

        26.4

        15

          
         

        P350/S993 (P346/S989)

        31.9

        15.2

          

        TMH6/TMH12

        F343/V982 (F339/V978)

        18.1

        16

        10

        [28]

         

        L339/V982 (L335/V978)

        17.1

        15.9

        16-25

         

        TMH6/TMH12

        L339/A985 (L335/A981)

        21.2

        16.8

        20-25

        [34]

         

        L332/L976 (L328/L972)

        14

        15.6

        9-13

         

        NBD/TMD

        L443/S909 (L439/S905)

        8.6

        12

        6-16

        [54]

         

        S474/R905 (S470/R905)

        10.1

        9.6

          

        NBD/TMD

        A266/F1086 (A262/F1082)

        10

        9.4

        5.5-15

        [55]

        WalkerA/Signature

        S1072/L531 (S1068/L527)

        62.4

        20.2

        5.5-15

        [56]

         

        S1072/S532 (S1068/L528)

        59.9

        18.5

          
         

        G1073/L531 (G1069/L527)

        65

        20.6

          
         

        G1073/L532 (G1069/L528)

        62.6

        19.3

          
         

        G1073/L533 (G1069/L529)

        62

        20.5

          
         

        C1074/L531 (C1070/L527)

        63.4

        21.8

          
         

        G1075/L531 (G1071/L527)

        64.8

        24.9

          
         

        S429/L1176 (S425/L1172)

        62.4

        25.1

          
         

        G430/L1176 (G426/L1172)

        65

        25.9

          
         

        C431/L1176 (C427/L1172)

        63.3

        28.3

          
         

        G432/L1176 (G428/L1172)

        64.9

        31.9

          

        Values from experimental cross-linking studies are derived from [41].

        Discussion

        Visualization of the molecular structures of human ABC transporters in 3D models contributes to the comprehension of the physical and chemical properties of these molecules, and of their intermolecular interactions with endogenous and exogenous molecules. Thus, interactions involved in determining the potencies and the specificities of different drugs with these drug targets can be identified. To construct a realistic molecular model ("target", e.g. human ABC transporters) by homology, based on an experimental structure ("template", e.g. the open inward facing Escherichia coli MsbA [19]), the sequence identity between the target and the template should be relatively high, and the target-template alignment should identify corresponding positions in the target and the template. Homology between two proteins indicates the presence of a common ancestor, and phylogenetic analyses of ABC transporters have indicated that eukaryotic ABCB transporters and ABCC transporters may have originated from bacterial multidrug transporters [47]. It has been shown that the homology modelling approach is at least as applicable to membrane proteins as it is to water-soluble proteins, and that sequence similarities of 30% between template and target will give a Cα-RMSD of 2 Å or less in TMHs [48]. The sequence identities between the template molecule MsbA and the target molecules ABCB1, ABCC4 and ABCC5 are 34%, 21% and 25%, respectively, and the secondary structure elements (NBDs and TMDs) are conserved. Sequence identities between the Escherichia coli MsbA TMD and the ABCB1, ABCC4 and ABCC5 TMD1s and TMD2s are 23% (ABCB1-TMD1), 21% (ABCB1-TMD2), 14% (ABCC4-TMD1), 18% (ABCC4-TMD2), 20% (ABCC5-TMD1) and 20% (ABCC5-TMD2), respectively.

        A multiple sequence T-COFFEE [38] alignment, which highlights evolutionary relationships and increases probability that corresponding sequence positions are correctly aligned, was used to create the target-template alignments in this study. The T-COFFEE [38] alignment differed from the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41] in TMH2 and TMH6. The ABCB1 model based on the combined alignment, with TMH2 adjusted corresponding to the previously published alignments of human ABCB1 and Escherichia coli MsbA [19, 41], was in the best agreement with cross-linking data and the X-ray crystal structure of the Mus musculus ABCB1 [20]. This illustrates that combining different alignment methods may strengthen the alignment used for homology modelling. The alignment correctly aligning TMH2 was created using ClustalW and HMMTOP [41], while T-COFFEE, which aligned TMH6 correctly, is broadly based on progressive approach to multiple alignment using a combination of local (Lalign) and global (ClustalW) pair-wise alignments to generate a library of alignment information which is used to guide the progressive alignment.

        The X-ray crystal structure of the Mus musculus ABCB1 [20] and site directed mutagenesis studies on ABCB1 may serve as validity tests both for helix orientation in the template [19], and for the alignment used for ABC transporter modelling (Figure 2). The helix orientation of the 12 TMHs of the ABCB1 model was in accordance with the X-ray crystal structure of the Mus musculus ABCB1 [20]. Both the ABCB1 model and the ABCB1 X-ray structure exhibited a V-shaped structure with the same relative domain orientations; TMDs twisted relative to the NBDs with TMH4 and TMH5 crossed over ("cross-over motif" [19]) and associated with TMD2, and TMH10 and TMH11 crossed over and associated with TMD1. The major difference between the ABCB1 model and the X-ray crystal structure of the Mus musculus ABCB1 [20] was that the V-shape of the ABCB1 model was wider than the X-ray crystal structure of the Mus musculus ABCB1 [20].

        Cα-Cα distances in the human ABCB1 model, Cα-Cα distances in the Mus musculus ABCB1 X-ray crystal structure [20], and distances between residues in the TMD area from experimental cross-linking studies on ABCB1, are listed in Table 3. As shown in the table, the Cα-Cα distances in the human ABCB1 model compared to the Cα-Cα distances in the Mus musculus ABCB1 X-ray crystal structure [20] revealed that the helix packing of TMH pairs 2 and 11, 5 and 11, 6 and 7, and 6 and 11, were only 1-2 Å further apart in the human ABCB1 model. TMHs 5 and 8 were packed approximately 1 Å tighter in the human ABCB1 model than in the Mus musculus ABCB1 X-ray crystal structure [20]. TMH pairs 1 and 11, 4 and 10, and 5 and 10 were approximately 3-7 Å further apart in the human ABCB1 model than in the Mus musculus ABCB1 X-ray crystal structure [20]. The most striking differences between helix packing of the human ABCB1 model and the Mus musculus ABCB1 X-ray crystal structure [20] were observed in TMHs 6 and 12. Whereas the differences of their packing towards other TMHs where in the range of 1-5 Å towards the extracellular side, the differences between the distances between these TMHs long in the human ABCB1 model and the Mus musculus ABCB1 X-ray crystal structure [20] were up to 15 Å towards the cytoplasm. This indicates that in order for ABCB1 to attain a wide open inward facing conformation, large conformational changes involving a scissors like movement of TMH6 and TMH12 may take place.

        As shown in Figure 5A, Ile306 (TMH5) [27, 35], Ile340 (TMH6) [33], Phe343 (TMH6) [21, 27], Phe728 (TMH7) [27], and Val982 (TMH12) [33, 35] may form a substrate recognition site in the ABCB1 model. The involvement of these amino acid residues is also confirmed by the X-ray crystal structure of the Mus musculus ABCB1 [20]. Interestingly, Ile306 (Ile302 in Mus musculus ABCB1) actually points slightly towards the membrane in the X-ray crystal structure, while it points directly towards the translocation pore in the ABCB1 model (Figure 5). This could be due to twisting of TMH5 upon changing conformation from at drug recognition conformation to a drug bound conformation. Cross-linking studies on ABCB1 has proposed that residue pairs Asn296-Gly774, Ile299-Phe770, Ile299-Gly774, and Gly300-Phe770 (TMH5 and TMH8, respectively), are adjacent [23]. These residues are in direct contact with each other in the ABCB1 model presented in this study. Furthermore, cross-linking studies has also shown that Val133 and Cys137 (TMH2) are close to Ala935 and Gly939 (TMH11) [24]. In the present ABCB1 model, these residues are adjacent. This also implies that the orientations of these residues in the models are correctly localized, and that the alignment used for the ICM homology modelling procedure is correct.

        As shown in Table 3, the Cα-Cα distances in the human ABCB1 model of residues that connect residues on both sides of the wings are substantially longer than distances measured by chemical cross-linking. This may be due to drug-induced fit in the cross-linking experiments, which is not reflected in the present open inward ABCB1 model. Interestingly, the corresponding Cα-Cα distances in the Mus musculus ABCB1 X-ray crystal structure [20] are also longer than distances measured by chemical cross-linking. The shorter distances measured by chemical cross-linking may represent conformations of ABCB1 that are closed to the cytoplasmic side, with the wings tighter than in the conformations of the human ABCB1 model and the Mus musculus ABCB1 X-ray crystal structure [20].

        The open inward facing Escherichia coli MsbA template may represent a functional inward-facing conformation of the transporter, even though conformational disruption of the protein due to the presence of detergent molecules during crystallization cannot be excluded. According to the Errat option of the SAVES Metaserver for analyzing and validating protein structures, which indicated that the stereochemical qualities of the models were realistic, the stereochemical quality of the template was poorer than the stereochemical qualities of the ABC transporter models (Table 1). This difference in quality may be due to the modelling procedures; the ABC transporter models were energy minimized using the AMBER 8.0 program package [37], whereas the template was not.

        Several ABCB1 models have previously been published [4952] based on an MsbA X-ray crystal structure that was subsequently retracted [53]. In 2009, 4 molecular models of human P-glycoprotein in two different catalytic states were published [41] based on X-ray crystal structures of the bacterial MsbA in different conformations [19]. These models are based on the previous alignments of human ABCB1 and Escherichia coli MsbA [19, 41], and consequently, the orientation of their TMH6 differ from the orientation of TMH6 in the ABCB1 model presented in this study. The measured Cα-Cα distances in our present ABCB1 model are in accordance with the corresponding distances in their open inward ABCB1 model [41].

        From a pharmacological point of view, the EPS of the ligand recognition area in the wide open conformation of each of the ABC transporters is of particular interest, since it may elucidate substrate differences between these transporters. The template structure was constructed by fitting the X-ray structure of outward facing MsbA to the electron density map of inward facing MsbA. The template conformation may therefore have limitations that can affect the calculated EPS in some regions of the models. ABCB1 transports cationic amphiphilic and lipophilic substrates [58], and, as illustrated in Figure 4, the EPS of its ligand recognition area was neutral with negative and weakly positive areas. In contrast, ABCC4 and ABCC5 transport organic anions [9], and the EPS of the ABCC5 substrate recognition area was generally positive. Interestingly, the substrate recognition area of ABCC4 was generally positive with negative area "spots". This may raise reflections over differences in substrate selectivity between the anionic transporters ABCC4 and ABCC5, and support the reports of ABCC4 with preference for cAMP and ABCC5 with preference for cGMP [9, 10]. The EPS of cAMP and cGMP (Figure 6) indicates that the surface of cGMP (Figure 6, panel B and D) has a larger region of negative EPS than that of cAMP (Figure 6, panel A and C). This may indicate that cGMP binds stronger to the surface of ABCC5 than cAMP, while negative area "spots" on the surface of ABCC4 may contribute to stronger binding to cAMP than to cGMP.
        http://static-content.springer.com/image/art%3A10.1186%2F1742-4682-6-20/MediaObjects/12976_2009_Article_202_Fig6_HTML.jpg
        Figure 6

        The surface of cAMP (panel A and C) and cGMP (panel B and D) colour coded according to electrostatic potentials outside the surface. The surface of cAMP in panel C are flipped 180° along the y-axis relative to panel A, while the surface of cGMP in panel D are flipped 180° along the y-axis compared with panel B.

        The residues of the binding site of the ligand bound Mus musculus ABCB1 X-ray crystal structure [20] and the respective binding site of all three models are shown in Table 2. While the binding sites of human and Mus musculus ABCB1 features lipophilic residues (Leucine, isoleucines, phenyl alanines, valine), ABCC4 has charged and polar residues and ABCC5 has polar residues. A positively charged residue in the binding site area of ABCC5, Lys448, also may take part in interaction with organic anions. The binding sites of the ABCB1, ABCC4 and ABCC5 models are wider and more accessible to the cytoplasm than the binding site of the Mus musculus ABCB1 X-ray crystal structure [20], reflecting their wide open-inward conformation.

        Crystal structures of ABC transporters captured in different conformations have revealed that ABC transporter mechanism involves alternating access of substrate from the inward to the outward facing conformation, with subunit twisting and domain swapping [1820]. The putative substrate recognition pocket in the ABCB1, ABCC4 and ABCC5 models in the wide open inward conformation presented in this study contains the same amino acid residues as the putative substrate releasing pocket in our previous outward-facing molecular models of ABCB1 [15], ABCC4 [16] and ABCC5 [17] based on the Staphylococcus aureus ABC transporter Sav1866 [18]; Ile306 (TMH5) [27, 35], Ile340 (TMH6) [33], Phe343 (TMH6) [21, 27], Phe728 (TMH7) [27], and Val982 (TMH12) [33, 35]. This indicates that these residues contribute to a substrate translocation pore that changes conformation from a high affinity inward facing substrate recognition binding site to a low affinity outward facing substrate releasing pocket. Mutating the corresponding residues of ABCC4 and ABCC5 (Table 2) into the ABCB1 residues would be a valuable test of our models. The models indicate that these mutants may have substrate specificity more similar to that of wild type ABCB1. Leu65 (TMH1) [26], which is also suggested to take part in ligand binding, and is localized in the substrate releasing pocket in the outward facing ABCB1 model [15], is slightly distant from the core area of the ligand recognition site in the inward facing ABCB1 model. This amino acid may come into contact with the ligand upon conformational changes associated with ligand binding.

        The models presented in this study may represent a substrate recognition conformation, and from a structure aided drug design point of view, the specificity and affinity of ABC transporter substrate binding in this conformation is of particular interest. When performing docking studies, the structural flexibility of transporters, and the structural changes of the drug and the drug target adopting an energetically favourable complex (induced-fit), as has been demonstrated in a cysteine-scanning mutagenesis and oxidative cross-linking study of substrate-induced changes in ABCB1 [22], should be considered in order to predict how a designed drug will fit into the drug target.

        The ABCB1, ABCC4 and ABCC5 models presented in this study should be considered as working tools for generating hypotheses and designing further experimental studies related to ABC transporter structure and function, and their drug interactions. The binding site of the ABCB1 transporter model is in accordance with X-ray crystal structure of the Mus musculus ABCB1 [20] and site directed mutagenesis data and cross-linking studies on ABCB1 [2135], indicating that the open inward-facing conformation structure of Escherichia coli MsbA [19] is a suitable template for homology modelling of ABCB1, ABCC4 and ABCC5. The corresponding residues in ABCC4 and ABCC5 (Table 2) are candidates for point mutations in site directed mutagenesis studies.

        Co-ordinates of the ABCB1, ABCC4 and ABCC5 models are available from the authors upon request.

        Declarations

        Acknowledgements

        We are grateful to Dr. Geoffrey Chang for providing us with the model of the open inward-facing conformation of the Escherichia coli MsbA X-ray crystal structure. This work was supported with grants from the Norwegian Cancer Society.

        Authors’ Affiliations

        (1)
        Department of Medical Pharmacology and Toxicology, Institute of Medical Biology, Faculty of Health Sciences, University of Tromsø

        References

        1. Saier MH: A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev. 2000, 64: 354-411. 10.1128/MMBR.64.2.354-411.2000.PubMed CentralView ArticlePubMed
        2. Higgins CF, Linton KJ: Structural biology. The xyz of ABC transporters. Science. 2001, 293: 1782-1784. 10.1126/science.1065588.View ArticlePubMed
        3. Oswald C, Holland IB, L S: The motor domains of ABC-transporters - What can structures tell us?. Naunyn-Schmiedeberg's Arch Pharmacol. 2006, 372: 385-399. 10.1007/s00210-005-0031-4.View Article
        4. Dean M, Rzhetsky A, Allikmets R: The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 2001, 11: 1156-1166. 10.1101/gr.GR-1649R.View ArticlePubMed
        5. Muller M, Mayer R, Hero U, Keppler D: ATP-dependent transport of amphiphilic cations across the hepatocyte canalicular membrane mediated by mdr1 P-glycoprotein. FEBS Lett. 1994, 343: 168-172. 10.1016/0014-5793(94)80312-9.View ArticlePubMed
        6. Orlowski S, Garrigos M: Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs. Anticancer Res. 1999, 19: 3109-3123.PubMed
        7. Smit JW, Duin E, Steen H, Oosting R, Roggeveld J, Meijer DK: Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level. Br J Pharmacol. 1998, 123: 361-370. 10.1038/sj.bjp.0701606.PubMed CentralView ArticlePubMed
        8. Wang EJ, Lew K, Casciano CN, Clement RP, Johnson WW: Interaction of common azole antifungals with P glycoprotein. Antimicrob Agents Chemother. 2002, 46: 160-165. 10.1128/AAC.46.1.160-165.2002.PubMed CentralView ArticlePubMed
        9. Borst P, de Wolf C, Wetering van de K: Multidrug resistance-associated proteins 3, 4, and 5. Pflugers Arch. 2007, 453: 661-673. 10.1007/s00424-006-0054-9.View ArticlePubMed
        10. Jedlitschky G, Burchell B, Keppler D: The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem. 2000, 275: 30069-30074. 10.1074/jbc.M005463200.View ArticlePubMed
        11. Dantzig AH, de Alwis DP, Burgess M: Considerations in the design and development of transport inhibitors as adjuncts to drug therapy. Adv Drug Deliv Rev. 2003, 55: 133-150. 10.1016/S0169-409X(02)00175-8.View ArticlePubMed
        12. Juliano RL, Ling V: A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976, 455: 152-162. 10.1016/0005-2736(76)90160-7.View ArticlePubMed
        13. Sampath J, Adachi M, Hatse S, Naesens L, Balzarini J, Flatley RM, Matherly LH, Schuetz JD: Role of MRP4 and MRP5 in biology and chemotherapy. AAPS PharmSci. 2002, 4: E14-10.1208/ps040314.View ArticlePubMed
        14. Zhou SF, Wang LL, Di YM, Xue CC, Duan W, Li CG, Li Y: Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development. Curr Med Chem. 2008, 15: 1981-2039. 10.2174/092986708785132870.View ArticlePubMed
        15. Ravna AW, Sylte I, Sager G: Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5). Theor Biol Med Model. 2007, 4: 33-10.1186/1742-4682-4-33.PubMed CentralView ArticlePubMed
        16. Ravna AW, Sager G: Molecular model of the outward facing state of the human multidrug resistance protein 4 (MRP4/ABCC4). Bioorg Med Chem Lett. 2008, 18: 3481-3483. 10.1016/j.bmcl.2008.05.047.View ArticlePubMed
        17. Ravna AW, Sylte I, Sager G: A molecular model of a putative substrate releasing conformation of multidrug resistance protein 5 (MRP5). Eur J Med Chem. 2008, 43: 2557-2567. 10.1016/j.ejmech.2008.01.015.View ArticlePubMed
        18. Dawson RJ, Locher KP: Structure of a bacterial multidrug ABC transporter. Nature. 2006, 443: 180-185. 10.1038/nature05155.View ArticlePubMed
        19. Ward A, Reyes CL, Yu J, Roth CB, Chang G: Flexibility in the ABC transporter MsbA: Alternating access with a twist. Proc Natl Acad Sci USA. 2007, 104: 19005-19010. 10.1073/pnas.0709388104.PubMed CentralView ArticlePubMed
        20. Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL, Chang G: Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science. 2009, 323: 1718-1722. 10.1126/science.1168750.PubMed CentralView ArticlePubMed
        21. Loo TW, Bartlett MC, Clarke DM: Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. J Biol Chem. 2003, 278: 50136-50141. 10.1074/jbc.M310448200.View ArticlePubMed
        22. Loo TW, Bartlett MC, Clarke DM: Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding. J Biol Chem. 2003, 278: 13603-13606. 10.1074/jbc.C300073200.View ArticlePubMed
        23. Loo TW, Bartlett MC, Clarke DM: Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane. J Biol Chem. 2004, 279: 7692-7697. 10.1074/jbc.M311825200.View ArticlePubMed
        24. Loo TW, Bartlett MC, Clarke DM: Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein. J Biol Chem. 2004, 279: 18232-18238. 10.1074/jbc.M400229200.View ArticlePubMed
        25. Loo TW, Bartlett MC, Clarke DM: ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein. Biochemistry. 2005, 44: 10250-10258. 10.1021/bi050705j.View ArticlePubMed
        26. Loo TW, Bartlett MC, Clarke DM: Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket. Biochem J. 2006, 396: 537-545. 10.1042/BJ20060012.PubMed CentralView ArticlePubMed
        27. Loo TW, Bartlett MC, Clarke DM: Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket. Biochem J. 2006, 399: 351-359. 10.1042/BJ20060715.PubMed CentralView ArticlePubMed
        28. Loo TW, Bartlett MC, Clarke DM: Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments. Biochemistry. 2007, 46: 9328-9336. 10.1021/bi700837y.View ArticlePubMed
        29. Loo TW, Clarke DM: Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12. J Biol Chem. 1997, 272: 20986-20989. 10.1074/jbc.272.34.20986.View ArticlePubMed
        30. Loo TW, Clarke DM: The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis. J Biol Chem. 2000, 275: 5253-5256. 10.1074/jbc.275.8.5253.View ArticlePubMed
        31. Loo TW, Clarke DM: Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil. J Biol Chem. 2001, 276: 14972-14979. 10.1074/jbc.M100407200.View ArticlePubMed
        32. Loo TW, Clarke DM: Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers. J Biol Chem. 2001, 276: 36877-36880. 10.1074/jbc.C100467200.View ArticlePubMed
        33. Loo TW, Clarke DM: Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein. J Biol Chem. 2002, 277: 44332-44338. 10.1074/jbc.M208433200.View ArticlePubMed
        34. Loo TW, Clarke DM: Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site. Proc Natl Acad Sci USA. 2002, 99: 3511-3516. 10.1073/pnas.022049799.PubMed CentralView ArticlePubMed
        35. Loo TW, Clarke DM: Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux. J Membr Biol. 2005, 206: 173-185. 10.1007/s00232-005-0792-1.View ArticlePubMed
        36. Abagyan R, Totrov M, Kuznetsov DN: ICM - a new method for protein modeling and design. Applications to docking and structure prediction from the distorted native comformation. J Comp Chem. 1994, 15: 488-506. 10.1002/jcc.540150503.View Article
        37. Case DA, Darden TA, Cheatham TE, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Wang B, Pearlman DA: AMBER 8. 2004, San Francisco: University of California
        38. Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217. 10.1006/jmbi.2000.4042.View ArticlePubMed
        39. Rost B, Yachdav G, Liu J: The PredictProtein server. Nucleic Acids Res. 2004, 32: W321-326. 10.1093/nar/gkh377.PubMed CentralView ArticlePubMed
        40. Bairoch A, Apweiler R: The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Res. 1999, 27: 49-54. 10.1093/nar/27.1.49.PubMed CentralView ArticlePubMed
        41. Becker JP, Depret G, Van Bambeke F, Tulkens PM, Prevost M: Molecular models of human P-glycoprotein in two different catalytic states. BMC Struct Biol. 2009, 9: 3-10.1186/1472-6807-9-3.PubMed CentralView ArticlePubMed
        42. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE: The Protein Data Bank. Nucleic Acids Res. 2000, 28: 235-242. 10.1093/nar/28.1.235.PubMed CentralView ArticlePubMed
        43. Abagyan R, Totrov M: Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J Mol Biol. 1994, 235: 983-1002. 10.1006/jmbi.1994.1052.View ArticlePubMed
        44. Laskoswki RA, MacArthur MW, Moss DS, Thorton JM: PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993, 26: 283-291. 10.1107/S0021889892009944.View Article
        45. Hooft RW, Vriend G, Sander C, Abola EE: Errors in protein structures. Nature. 1996, 381: 272-10.1038/381272a0.View ArticlePubMed
        46. Colovos C, Yeates TO: Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 1993, 2: 1511-1519. 10.1002/pro.5560020916.PubMed CentralView ArticlePubMed
        47. Igarashi Y, Aoki KF, Mamitsuka H, Kuma K, Kanehisa M: The evolutionary repertoires of the eukaryotic-type ABC transporters in terms of the phylogeny of ATP-binding domains in eukaryotes and prokaryotes. Mol Biol Evol. 2004, 21: 2149-2160. 10.1093/molbev/msh226.View ArticlePubMed
        48. Forrest LR, Tang CL, Honig B: On the accuracy of homology modeling and sequence alignment methods applied to membrane proteins. Biophys J. 2006, 91: 508-517. 10.1529/biophysj.106.082313.PubMed CentralView ArticlePubMed
        49. Vandevuer S, Van Bambeke F, Tulkens PM, Prevost M: Predicting the three-dimensional structure of human P-glycoprotein in absence of ATP by computational techniques embodying crosslinking data: Insight into the mechanism of ligand migration and binding sites. Proteins. 2006, 63: 466-478. 10.1002/prot.20892.View ArticlePubMed
        50. Seigneuret M, Garnier-Suillerot A: A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure. J Biol Chem. 2003, 278: 30115-30124. 10.1074/jbc.M302443200.View ArticlePubMed
        51. Stenham DR, Campbell JD, Sansom MS, Higgins CF, Kerr ID, Linton KJ: An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling. Faseb J. 2003, 17: 2287-2289.PubMed
        52. Shilling RA, Balakrishnan L, Shahi S, Venter H, van Veen HW: A new dimer interface for an ABC transporter. Int J Antimicrob Agents. 2003, 22: 200-204. 10.1016/S0924-8579(03)00212-7.View ArticlePubMed
        53. Chang G, Roth CB, Reyes CL, Pornillos O, Chen YJ, Chen AP: Retraction. Science. 2006, 314: 1875-10.1126/science.314.5807.1875b.View ArticlePubMed
        54. Zolnerciks JK, Wooding C, Linton KJ: Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein. Faseb J. 2007, 21: 3937-3948. 10.1096/fj.07-8610com.View ArticlePubMed
        55. Loo TW, Bartlett MC, Clarke DM: Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR). J Biol Chem. 2008, 283: 28190-28197. 10.1074/jbc.M805834200.PubMed CentralView ArticlePubMed
        56. Loo TW, Bartlett MC, Clarke DM: The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. J Biol Chem. 2002, 277: 41303-41306. 10.1074/jbc.C200484200.View ArticlePubMed

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        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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