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Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5).

Ravna AW, Sylte I, Sager G - Theor Biol Med Model (2007)

Bottom Line: In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model.The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5.The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5.

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Affiliation: Department of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway. Aina.W.Ravna@fagmed.uit.no

ABSTRACT

Background: Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette) transporters human P-glycoprotein (ABCB1) and the human MRP5 (ABCC5) are involved in multidrug resistance.

Results: In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS) of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1)), Ile306 (TMH5), Ile340 (TMH6) and Phe343 (TMH6) may form a binding site, and this is in accordance with previous site directed mutagenesis studies.

Conclusion: The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5.

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Alignment. Alignment of VC-Sav1866, ABCB1 and ABCC5 used as input alignment for the ICM homology modeling module. TMHs are indicated in red boxes, Walker A motifs are indicated in yellow boxes, and Walker B motifs are indicated in green boxes.
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Figure 5: Alignment. Alignment of VC-Sav1866, ABCB1 and ABCC5 used as input alignment for the ICM homology modeling module. TMHs are indicated in red boxes, Walker A motifs are indicated in yellow boxes, and Walker B motifs are indicated in green boxes.

Mentions: The crystal structure of Sav1866 [23] (pdb code 2HYD), which has a 3 Å resolution, was used as template to construct a homology model of ABCB1 (Swiss-Prot accession code P08183), using the ICM software versions 3.4–9b [47]. T-COFFEE, Version 4.71 available at the Le Centre national de la recherche scientifiquewebsite [49], and ICM version 3.4–4 [47], were used to create multiple sequence alignments of human ABCB1, human ABCC5, human ABCC11 (SWISS-PROT accession number Q9BX80), human ABCC4 (SWISS-PROT accession number O15439), Sav1866 (SWISS-PROT accession number Q99T13), Vibrio cholerae MsbA (SWISS-PROT accession number Q9KQW9) and Escherichia coli MsbA (SWISS-PROT accession number P60752). The alignments were used as a basis, and adjusted in ICM for gaps for the input alignment in the ICM homology modeling module. To strengthen the sequence alignment, secondary structure predictions were performed to define the boundaries of the TMHs using the PredictProtein server for sequence analysis and structure prediction [50], and SWISS-PROT [46]. The alignment of Sav1866, ABCB1 and ABCC5 is shown in Figure 5. The ICM homology modeling module constructs the model from a few core sections defined by the average of Cα atom positions in the conserved regions. Loops are constructed by searching within thousands of high quality structures in the PDB databank [51] by matching them in regard to sequence similarity and sterical interactions with the surroundings of the model. The best fitting loops are selected based on their relative energies. N- and C-terminals were not included in the models. Because of the length of the loop connecting NBD1 and TMD2, the loop was particularly difficult to model. In the generated models of the loop, the residues had a tendency to overlap with surrounding amino acids (sterical clashes), and more than 20 models was constructed before a model without sterical clashes was generated. In order to accomplish this, the start of the loop was moved one amino acid further towards the N-terminal direction, or the end of the loop was moved one amino acid further towards the C-terminal direction, in the ICM input alignment per modeling round, making the input loop longer until a model with no sterical clashes was generated. The alignment shown in figure 5 is the exact input alignment used for the final model. The construction of the ABCC5 model is described in a previous cGMP docking study (submitted).


Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5).

Ravna AW, Sylte I, Sager G - Theor Biol Med Model (2007)

Alignment. Alignment of VC-Sav1866, ABCB1 and ABCC5 used as input alignment for the ICM homology modeling module. TMHs are indicated in red boxes, Walker A motifs are indicated in yellow boxes, and Walker B motifs are indicated in green boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211457&req=5

Figure 5: Alignment. Alignment of VC-Sav1866, ABCB1 and ABCC5 used as input alignment for the ICM homology modeling module. TMHs are indicated in red boxes, Walker A motifs are indicated in yellow boxes, and Walker B motifs are indicated in green boxes.
Mentions: The crystal structure of Sav1866 [23] (pdb code 2HYD), which has a 3 Å resolution, was used as template to construct a homology model of ABCB1 (Swiss-Prot accession code P08183), using the ICM software versions 3.4–9b [47]. T-COFFEE, Version 4.71 available at the Le Centre national de la recherche scientifiquewebsite [49], and ICM version 3.4–4 [47], were used to create multiple sequence alignments of human ABCB1, human ABCC5, human ABCC11 (SWISS-PROT accession number Q9BX80), human ABCC4 (SWISS-PROT accession number O15439), Sav1866 (SWISS-PROT accession number Q99T13), Vibrio cholerae MsbA (SWISS-PROT accession number Q9KQW9) and Escherichia coli MsbA (SWISS-PROT accession number P60752). The alignments were used as a basis, and adjusted in ICM for gaps for the input alignment in the ICM homology modeling module. To strengthen the sequence alignment, secondary structure predictions were performed to define the boundaries of the TMHs using the PredictProtein server for sequence analysis and structure prediction [50], and SWISS-PROT [46]. The alignment of Sav1866, ABCB1 and ABCC5 is shown in Figure 5. The ICM homology modeling module constructs the model from a few core sections defined by the average of Cα atom positions in the conserved regions. Loops are constructed by searching within thousands of high quality structures in the PDB databank [51] by matching them in regard to sequence similarity and sterical interactions with the surroundings of the model. The best fitting loops are selected based on their relative energies. N- and C-terminals were not included in the models. Because of the length of the loop connecting NBD1 and TMD2, the loop was particularly difficult to model. In the generated models of the loop, the residues had a tendency to overlap with surrounding amino acids (sterical clashes), and more than 20 models was constructed before a model without sterical clashes was generated. In order to accomplish this, the start of the loop was moved one amino acid further towards the N-terminal direction, or the end of the loop was moved one amino acid further towards the C-terminal direction, in the ICM input alignment per modeling round, making the input loop longer until a model with no sterical clashes was generated. The alignment shown in figure 5 is the exact input alignment used for the final model. The construction of the ABCC5 model is described in a previous cGMP docking study (submitted).

Bottom Line: In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model.The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5.The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway. Aina.W.Ravna@fagmed.uit.no

ABSTRACT

Background: Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette) transporters human P-glycoprotein (ABCB1) and the human MRP5 (ABCC5) are involved in multidrug resistance.

Results: In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS) of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1)), Ile306 (TMH5), Ile340 (TMH6) and Phe343 (TMH6) may form a binding site, and this is in accordance with previous site directed mutagenesis studies.

Conclusion: The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5.

Show MeSH