Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1)

Abstract

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

Figure 1. Inhibition of photocrosslinking of mutant Pgps with IAAP by cyclosporine A and tariquidar.

Inhibition of IAAP labeling for single mutants Q725C, Y307C, F728C and V982C (upper graphs) and for double Q725C/V982C, Y307C/V982C, F728C/V982C, and triple Y307C/Q725C/V982C (lower graphs) mutants at different concentrations of (A) CsA and (B) tariquidar, are shown. Inhibition of IAAP labeling for cysless WT is included in all graphs, as a reference. Autoradiograms corresponding to cysless, V982C and Y307C/Q725C/V982C, as representative examples of complete inhibition, partial inhibition and no inhibition of IAAP-labeling, respectively, are shown at the top of the figure. Crude membranes containing Pgp (60-80 µg protein) were treated with increasing concentrations of CsA and tariquidar in 100 µL buffer containing 50 mM MES-Tris pH 6.8 for 10 min at 37°C. Then, samples were photocrosslinked with IAAP at 4°C as described in the Materials and Methods section. Mean values from at least three independent experiments are plotted and error bars depict SD.

Figure 2. Some of the drug-binding pocket mutants exhibit very low basal ATPase activity.

(A) The expression of mutant Pgps in High-Five insect cell membranes. Crude membranes of High-Five insect cells expressing indicated mutant Pgps were analyzed by standard SDS-PAGE method and proteins were stained with Colloidal Blue staining. Each lane contains 20 µg total membrane protein. Only the portion of the 7% Tris-Acetate gel with the Pgp band is shown. Expression level of mutant Pgps was quantified and compared to cysless WT Pgp using ImageJ 1.46r. (B) Basal ATP hydrolysis (in the absence of any added compound) of cysless WT and mutant Pgps is shown in bar graph format. The mutants are ordered from the lowest to the highest basal activity. Vanadate-sensitive Pgp-mediated ATP hydrolysis was measured as described in Materials and Methods. In all experiments, mutant Pgps (10-25 µg protein/100 µL) were incubated at 37°C and the ATPase activity was measured after 20 min reaction, in the presence of 5 mM ATP. At least three experiments were carried out for each mutant and error bars represent SD.

Figure 3. Modulation of ATP hydrolysis by drugs is retained by mutant Pgps.

(A) Stimulation of ATP hydrolysis by verapamil (50 µM), QZ59-SSS (1 µM), valinomycin (10 µM) and FSBA (1 mM) are shown in bar graph format; basal activity (in the absence of added drug) indicated by dashed line is equal to one. (B) Inhibition of verapamil-stimulated ATP hydrolysis by CsA (10 µM) and tariquidar (5 µM) is shown. The ATPase activity is measured in the presence of verapamil (5 µM) because of the low basal activity of most of the mutants; the first bar thus indicates the activity in the presence of 5 µM verapamil, which is taken as 100% (control). Vanadate-sensitive Pgp-mediated ATP hydrolysis was measured as described in Materials and Methods. In the case of FSBA, 5 mM ATP was added prior to the addition of FSBA [21]. At least two independent experiments were carried out for each mutant with indicated compounds. Additional information including standard deviations, fold-stimulation or % inhibition, and number of experiments is given in the section (Tables S2 and S3).

Figure 4. BacMam-baculovirus-transduced HeLa cells express normal levels of mutant Pgps at the cell surface.

(A) The left panel shows the cell surface localization of Y307C with human Pgp-specific monoclonal antibody MRK-16 labeling as detected by green fluorescence detector. The middle panel shows the same for the double mutant Y307C/V982C, and the right panel for the triple mutant Y307C/Q725C/V982C. (B) The panels show the detection of the mutant Pgps with conformation-sensitive monoclonal UIC2 antibody after the treatment of cells with 20 µM CsA for 5 min at 37°C as described in Materials and Methods. The conformation sensitivity towards CsA of single (Y307C), double (Y307C/V982C) and triple (Y307C/Q725C/V982C) mutants was similar to cysless WT Pgp, as shown in the three panels, respectively. The histograms in both (A) and (B) are from one representative experiment, which was done independently at least three times. Samples treated with IgG2a isotype antibody are labeled as control. In all panels, the level of expression of cysless WT Pgp is shown for comparison and it is taken as 100% (see Table 2).

Figure 5. The transport functions of single (Y307C), double (Y307/V982C) and triple (Y307C/Q725C/V982C) mutants are differentially affected.

(A) Rhodamine 123 and (B) Bodipy-FL-prazosin accumulation in HeLa cells. The inactive E501Q/E1201Q (EQ) mutant P-gp is used as a control to show completely aborted function as described in Materials and Methods. The HeLa cells expressing mutant-Pgps were incubated with either rhodamine 123 (1.3 µM) or bodipy-FL-prazosin (0.5 µM) for 45 min at 37°C. The cells were washed and subsequently analyzed by flow cytometry as described in Materials and Methods. The histograms show results from a typical experiment. Similar results were obtained in at least 3-4 experiments. The transport function of all mutants with six fluorescent substrates is summarized in Table 2.

Figure 6. Docking of cyclosporine A, tariquidar, valinomycin and FSBA in the binding pocket of human Pgp.

The homology model of human Pgp was generated using the recently described structure of mouse Pgp as a template (4KSB.pdb) [32]. Exhaustive ligand docking in the homology model of human Pgp was carried out using receptor grids centered at the position of the QZ59-RRR molecule (shown as red sticks), with flexible side-chains covering an ample space and a search box of dimensions 40Å × 35Å × 35Å. The first 10 modes with the highest docking scores were clustered and shown as black stick models for each drug. Docking results suggest different sites depending on the size of the molecule. QZ59-RRR (red) and QZ59-SSS (blue) molecules are included as stick models in their original positions, as indicated by the crystal structures 3G60.pdb and 3G61.pdb, respectively. Alpha-helices 4 and 5 are not shown for clarity; the rest of the helices of transmembrane domain 1 (green) and transmembrane domain 2 (cyan) are labeled and shown as cartoon models. The figure was prepared with PYMOL.