Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

Abstract

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC₅₀ values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.

Keywords: ABC transporter; ATP hydrolysis; P-glycoprotein; detergent micelles; docking; drug resistance; molecular modeling; multidrug transporter; nanodiscs; proteoliposomes.

Figure 1.

Effect of tariquidar, elacridar, or zosuquidar on ATP hydrolysis by mP-gp depends on its presence in either native membranes or detergent micelles. Mouse P-gp-expressing native membranes of High-Five insect cells (50–100 μg of protein/ml; panels A-C) or purified P-gp in gel filtration buffer (1.5–3 μg of protein; panels D–F) was incubated with the indicated concentrations of tariquidar (A and D), elacridar (B and E), and zosuquidar (C and F) at 37 °C for 5 min. The ATP hydrolysis was then determined by adding 5 mm ATP as described under “Experimental procedures.” The basal specific ATPase activity in native membranes (for panels A–C) and in detergent micelles (for panels D–F) ranged from 42 to 54 and 79 to 83 nmol of P_i/min/mg of protein, respectively. The basal activity in the presence of DMSO alone was subtracted, and the percent stimulation or inhibition of ATP hydrolysis (y axis) was plotted as a function of varying concentrations of the given modulator (x axis). The curves were fitted using GraphPad Prism 7.0 with nonlinear one-phase exponential decay or association analysis. The curves represent the mean ± S.D. from three independent experiments. The IC₅₀ and EC₅₀ values derived from the curves are given in Table 1.

Figure 2.

Effect of verapamil and QZ59-SSS on the ATP hydrolysis of P-gp in native membranes, detergent micelles, or proteoliposomes. Mouse P-gp-expressing membranes from High-Five insect cells (A and D), concentrated purified mP-gp in gel filtration buffer (detergent micelles) (B and E), or proteoliposomes reconstituted with purified P-gp (C and F) were incubated with the indicated concentrations of verapamil (A–C) and QZ59-SSS (D–F) at 37 °C for 5 min. The ATP hydrolysis was then initiated by adding 5 mm ATP as described under “Experimental procedures.” The basal-specific ATPase activity in native membranes (panels A and D) and in detergent micelles (panels B and E) and proteoliposomes (panels C and F) ranged from 42 to 54, 79 to 83, and 716 to 730 nmole of P_i/min/mg of protein, respectively. The percent stimulation of the ATP hydrolysis (y axis) was plotted as a function of varying concentrations of the compound (x axis). The curves were fitted using GraphPad Prism version 7.0 with nonlinear one-phase association analysis. The curves represent the mean ± S.D. values from three independent experiments. The EC₅₀ values derived from the curves are given in Table 1.

Figure 3.

Tariquidar's ability to inhibit photocross-linking of mP-gp with IAAP was significantly decreased in detergent micelles. Native membranes from mP-gp-expressing High-Five insect cells (25–50 μg of protein) (A) or purified mP-gp (2–5 μg of protein) (B) were incubated with increasing concentrations of tariquidar at room temperature for 5 min followed by photocross-linking with ¹²⁵I-AAP (5 nm) using 366-nm UV light as described under “Experimental procedures.” The photocross-linked P-gp samples were separated on 7% Tris acetate SDS-PAGE gels. The gels were dried and exposed to X-ray films. Representative autoradiograms of IAAP-labeled P-gp bands in native membranes (panel A) and detergent micelles (panel B) are shown at the top. The concentration of tariquidar (Tar) is given above each lane. The curves of IAAP incorporation (% of control) as a function of tariquidar concentration were plotted using GraphPad Prism 7.0 with nonlinear one-phase exponential decay analysis. Similar results were obtained in three independent experiments.

Figure 4.

Effect of tariquidar (A), elacridar (B), and zosuquidar (C) on purified hP-gp-mediated ATP hydrolysis. Purified and concentrated hP-gp in gel filtration buffer with ∼67.5× CMC DDM (2.5–3.5 μg of protein) was incubated with the indicated concentrations of the inhibitors at 37 °C for 5 min. ATP hydrolysis mediated by purified hP-gp in the presence of these modulators was determined as described under “Experimental procedures.” The basal specific ATPase activity was in the range of 70–80 nmole of P_i/min/mg of protein. The analysis was done as described in the legend to Fig. 1. Shown here are graphs with mean ± S.D. values of three independent experiments. The EC₅₀ values derived from the curves were 6.68 ± 1.32, 8.86 ± 1.53, and 10.22 ± 2.75 μm for tariquidar, elacridar, and zosuquidar, respectively.

Figure 5.

Effect of the addition of excess E. coli total phospholipids to purified protein in a detergent buffer on ATP hydrolysis mediated by mP-gp and hP-gp. Sonicated E. coli total phospholipids were added to purified mP-gp (A) or hP-gp (B) in a 25:1 w/w ratio to purified P-gp in gel filtration buffer. The reaction mixture was incubated at 37 °C for 10 min followed by the addition of 10 μm tariquidar, elacridar, and zosuquidar as indicated. The ATP hydrolysis was evaluated as described under “Experimental procedures.” The histograms show the ATPase-specific activity (y axis) in the presence of these modulators (x axis). Mean ± S.D. values from three independent experiments are given.

Figure 6.

Interaction of mP-gp with inhibitors in proteoliposomes is similar to that in native membranes. A and B, tariquidar and elacridar inhibit basal ATPase activity of purified mP-gp after reconstitution in proteoliposomes. Purified mP-gp was reconstituted into proteoliposomes using E. coli total phospholipids as described under “Experimental procedures.” Proteoliposomes containing 2 μg of purified P-gp were incubated with indicated concentrations of tariquidar (A) or elacridar (B), and the ATP hydrolysis was measured as described under “Experimental procedures.” The specific basal ATP activity ranged from 716 to 730 nanomoles P_i/min/mg of protein. The percent inhibition of basal activity was plotted (y axis) as a function of concentration of the inhibitors (x axis) as described in the legend to Fig. 1. Values shown represent the mean ± S.D. from at least three independent experiments. The IC₅₀ values derived from the curves are given in Table 1. C, inhibition of IAAP incorporation in P-gp by tariquidar. Proteoliposomes reconstituted with mP-gp (2.5–5 μg of protein) were incubated with DMSO (−) or 1 μm tariquidar (+) for 5 min at room temperature. The samples were photocross-linked with IAAP and separated on a 7% Tris acetate SDS-PAGE gel, and the autoradiogram was developed as described in the legend to Fig. 3. Shown here is a representative autoradiogram from three independent experiments.

Figure 7.

Effect of tariquidar and elacridar on the ATPase activity of mP-gp reconstituted in nanodiscs. A, SDS-PAGE of purified mP-gp in DDM micelles and after its reconstitution in nanodiscs. mP-gp-containing nanodiscs with membrane scaffold protein (MSP1E3D1) were prepared as described under “Experimental procedures.” Purified mP-gp in DDM micelles (lane 1; 0.5 μg of protein) and in nanodiscs (lane 2; 0.5 μg of protein) was resolved on 7% Tris acetate gel and stained with InstantBlue (Coomassie-based sensitive stain was from Expedeon Inc., San Diego, CA). Please note that P-gp and MSP, due to their hydrophobic nature, travel to lower positions instead of traveling to the normal positions corresponding to their true molecular sizes. B and C, ATP hydrolysis mediated by mP-gp in these nanodiscs was measured in the presence of indicated concentration of tariquidar (B) and elacridar (C) as described under “Experimental procedures.” The basal ATPase activity in nanodiscs was in the range of 298–322 nanomoles of P_i/min/mg of protein. The percent inhibition of ATP hydrolysis (considering basal activity as 100%) and IC ₅₀ values were calculated as described in the legend to Fig. 1. The IC₅₀ values for inhibition by tariquidar and elacridar were 1.55 ± 0.38 and 1.18 ± 0.22 μm, respectively.

Figure 8.

Docking of tariquidar and DDM in the binding pocket of P-gp. Exhaustive ligand docking in a homology model of hP-gp based on mP-gp structure 4M2T.pdb (A) and in the mP-gp structure 4Q9H.pdb (B) was carried out using the AutoDock Vina program with a receptor grid centered at the position of the QZ59-RRR molecule, with flexible side chains (26 residues) and a search box of dimensions 40 Å × 35 Å × 35 Å. The structure 4Q9H was previously aligned to 4M2T to use the same receptor grid in both cases. The first 10 modes with the highest docking scores (given in the figure) were clustered and shown as blue (tariquidar) and magenta (DDM) stick models. N-terminal and C-terminal domains are shown in different colors (green and gray for PDB code 4M2T and light green and gray for 4Q9H, respectively). TM helices 10 and 12 are not shown for clarity. The figure was prepared with PyMOL 1.4.1.

Figure 9.

Schematic of binding of tariquidar in the substrate-binding pocket of P-gp in membranes and detergent micelles. A, representation of tariquidar binding that leads to inhibition of the basal ATPase activity of P-gp in native membranes. The figure shows the docking of tariquidar in a homology model of hP-gp based on mP-gp X-ray structure 4M2T (4). B, schematic representation of the binding of tariquidar at a different site when the preferred site is occupied by DDM. Based on cryo-EM data, the DDM molecules surround the hydrophobic transmembrane area of P-gp (13). The consequent binding of tariquidar to another (low-affinity) site leads to stimulation of the basal ATP hydrolysis of P-gp in detergent micelles (Fig. 1D). The residues lining this site are not yet known. When P-gp is reconstituted in proteoliposomes, tariquidar binding leads to inhibition of the basal ATP hydrolysis similar to that observed with native membranes (panel A). The N-terminal half of P-gp is shown in green, and the C-terminal half is in gray. Transmembrane helices 10 and 12 were removed to better visualize the substrate-binding pocket. Tariquidar (blue) and DDM (magenta) are shown as spheres. The position of the lipid bilayer (insect cell membrane in A) is determined by the visible hydrophobic region of the protein surface. The structures were created with PyMOL version 1.4.1.