Effect of Detergent Micelle Environment on P-glycoprotein (ABCB1)-Ligand Interactions
Abstract
P-glycoprotein (P-gp) is a multidrug transporter that utilizes energy from ATP hydrolysis to efflux a variety of structurally dissimilar hydrophobic and amphipathic compounds including anticancer drugs from cells. Several structural studies on purified P- gp have been reported and there is very limited and in some cases conflicting information available on ligand interactions with isolated transporter in a dodecyl maltoside detergent environment. In this report, we compare the biochemical properties of human and mouse P-gp in native membranes, detergent micelles, and after reconstitution 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, whereas these drugs inhibited the ATPase activity of the transporter in native membranes or when it was reconstituted in proteoliposomes, with IC50 values in the 10 to 40 nanomolar range. Similarly, a 30- to 150- fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59- SSS was observed in detergent micelles compared to native or artificial membranes. These findings in aggregate demonstrate that the high-affinity site is inaccessible either due to 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 recommend that structural and functional aspects of ligand interactions with purified P- gp and other ATP-Binding Cassette transporters that transport amphipathic or hydrophobic substrates be studied in a detergent-free native or artificial membrane environment.
P-glycoprotein (P-gp; ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily of membrane proteins, which is composed of forty-eight known membrane transporters divided into seven classes denoted “A” through “G” (1). P-gp is a member of the “B” class of ABC transporters, designated ABCB1 and is best characterized as a single polypeptide containing two symmetrical halves that are 65% homologous to one another (2,3). Each half is comprised of a transmembrane domain (TMD) containing six transmembrane helices and a cytoplasmic nucleotide-binding domain (NBD). The NBDs of P-gp consist of a catalytic core domain and an -helical subdomain specific to ABC transporters. The catalytic cores of NBD1 and NBD2 each contain a Walker A, Walker B, and signature C motif which mediates ATP hydrolysis by the transporter.We earlier observed that the interaction of modulators with their binding sites in the TMDs of human P-gp, as measured by the ligand’s effect on ATP hydrolysis and modulator binding, depends on their interactions with amino acids present in the substrate-binding pocket of the protein (4). Specifically, we discovered that certain drugs such as zosuquidar, elacridar and tariquidar that normally inhibit ATP hydrolysis mediated by human P-gp switch to stimulation of ATP hydrolysis when a pair of phenylalanine- tyrosine structural motifs in the substrate- binding pocket was mutated (4). Molecular modeling studies further led us to infer that formation of hydrogen bonds between these inhibitors and tyrosine residues of the structural motifs in the substrate-binding pocket determine the affinity of modulators.
Structural studies on P-gp by X-ray crystallography (5-10), EM (11,12) and with cryo-EM (13,14) have been carried out using purified protein in n-dodecyl β-D-maltoside (DDM) micelles. In a majority of cases, high concentrations of substrates or modulators had to be used to observe their effects on ATPase activity of purified transporter in detergent micelles. In addition, attempts to co-crystalize mouse or human P-gp in detergent solution with modulators including zosuquidar, tariquidar and elacridar, which exhibit very high affinity in the 10-40 nanomolar range in native membranes, have been unsuccessful.In the present study, we evaluated the effect of changing the normal lipid environment of purified P-gp to a detergent environment on the interaction of ligands and modulators with the transporter. We show that the detergent micelle environment used to purify the protein leads to a change from inhibition by high-affinity inhibitors to stimulation of ATP hydrolysis of P-gp due to their interaction at an alternate site with decreased affinity. Ligand binding affinity in the substrate-binding pocket is known to modulate the ATPase activity by influencing the release of ADP, a rate-limiting step in the catalytic cycle of P-gp (15). Similarly, there is a 30 to 150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS, which stimulate ATPase activity even in detergent micelles. Interestingly, addition of excess phospholipid to the detergent-protein mixture alone does not overcome the detergent effect, but removal of detergent with Bio-Beads SM-2 in the presence of excess phospholipids and generation of proteoliposomes restores the apparent affinities of ligands for the transporter to similar levels as observed in native membranes. These findings in aggregate demonstrate that due to inaccessibility of the high affinity site for interactions in a detergent micelle environment, ligands bind to a low affinity site, resulting in altered modulation of P-gp ATPase activity. We suggest that the structural and functional properties of ligand interactions with purified P-gp and other ABC transporters that transport amphipathic or hydrophobic substrates should be studied in a detergent-free artificial membrane (proteoliposomal or possibly nanodisc) environment.
Results
The detergent DDM was used for crystallization of mouse and C. elegans P-gp and the ATPase activity of P-gp was measured in DDM micelles in the presence of added lipid (16,17). However, to this date a systematic comparison of the effect of substrates and modulators on the ATPase activity of P-gp in native membranes, after its purification in DDM micelles and following reconstitution in proteoliposomes or nanodiscs has not been reported. In this report we characterize the ligand interactions with mouse and human P-gp (in the following sections, mouse P-gp is referred as mP-gp and human P-gp as hP-gp, respectively) in native membranes, and after its purification in DDM micelles and reconstitution in nanodiscs and proteoliposomes under similar conditions to those used for cryo-EM and generation of crystals for X-ray crystallography studies.Differential Modulation of ATPase Activity of P-gp by Tariquidar, Elacridar and Zosuquidar When Present in Native Membranes or Detergent Micelles—To determine the effect of changing the environment from membranes to detergent micelles, ATP hydrolysis by mP-gp in native membranes and after its purification in DDM micelles in the presence of three known and well- characterized inhibitors (tariquidar, elacridar and zosuquidar) was measured, as detailed in Experimental Procedures. It was observed that while all three drugs inhibited the basal (in the presence of DMSO solvent only) ATP hydrolysis mediated by mP-gp in native membranes with IC50 values in the low nanomolar range (Fig. 1, A-C), these inhibitors instead stimulated ATP hydrolysis of mP-gp in DDM detergent micelles in a concentration- dependent manner with EC50 values 0.51,2.18 and 11.82 µM (Fig. 1, D-F). In addition, we also evaluated the effect of verapamil and QZ59-SSS, two compounds which are known to stimulate ATP hydrolysis by P-gp (18) and (19), on the ATPase activity of purified mP-gp in DDM micelles. Both verapamil and QZ59- SSS also stimulated the ATP hydrolysis of mP- gp in native membranes. However, the EC50 values for stimulation of ATP hydrolysis were 150-fold and 30-fold higher in detergent micelles than in native membranes in the presence of verapamil (Fig. 2 A-B and QZ59- SSS (Fig. 2 D-EB), respectively. These data show that the affinity of compounds that stimulate the ATPase activity of P-gp is also decreased in detergent micelles.
Significant Decrease in the Ability of Tariquidar to Inhibit Photolabeling of Purified mP-gp with IAAP in Detergent Micelles— Based on the above data, it appeared that purified mP-gp in detergent micelles has properties which are different when the transporter is present in native membranes. There is a possibility that the conformation of the substrate-binding pocket of the protein is altered in a detergent environment, thereby resulting in a change of behavior when interacting with ligands. To further study this change, we evaluated the ability of tariquidar, a representative inhibitor from this class, to inhibit the binding of another ligand, 125Iodoarylazidoprazosin (IAAP), which is transported by P-gp (20). As shown in Fig. 3A, while pretreatment of the native membranes with tariquidar in a concentration-dependent manner inhibited the photolabeling of mP-gp with IAAP with IC50 0.1 ± 0.01 µM, there was only partial inhibition (40-45%) even at a 10 µM drug concentration of the binding of IAAP to purified mP-gp in DDM micelles (Fig. 3B), with apparent IC50 >10 µM. Thus, photolabeling with IAAP provided further evidence for the significantly decreased affinity of tariquidar for purified mP-gp in detergent micelles.Purified hP-gp in Detergent Micelles Has Similar ATP Hydrolysis Properties as mP-gp— As the above studies were done with mP-gp, we wanted to investigate its relevance to hP- gp, which shares 87% sequence identity and 93% sequence similarity with mouse mdr1a and has functional similarities. However, by X- ray crystallography, resolution of the structure of hP-gp has been proven to be a very challenging task. hP-gp was purified from High-Five cell membranes as described in Experimental Procedures in the presence of DDM, and the effect of the inhibitors tariquidar, elacridar and zosuquidar on its ATPase activity in DDM micelles was evaluated. Similar to mP-gp, these inhibitors also showed a concentration-dependent stimulation of ATP hydrolysis, with EC50 values of 6.68 (tariquidar), 8.86 (elacridar) and 10.22 (zosuquidar) μM, respectively (Fig. 4, A-C). Therefore, stimulation of ATP hydrolysis-mediated by hP-gp by these modulators in detergent micelles was the opposite effect as observed in native membranes (4). Stimulation of the ATPase activity of purified hP-gp up to 10-fold by tariquidar in DDM micelles in the presence of lipids was also observed earlier (21).
Addition of Excess Phospholipids to Purified P-gp in Detergent Micelles Does Not Change ATP Hydrolysis Properties—As stated above, when P-gp is in the presence of detergent micelles its interaction with inhibitors is altered, as well as its apparent affinity for verapamil and the cyclic peptide inhibitor QZ59-SSS. Therefore, we sought to determine whether replacing or diluting the detergent by adding excess phospholipids would lead to a change in its biochemical properties. In this regard, sonicated E. coli total phospholipid mixture was added to purified mP-gp and hP- gp in excess concentration (P-gp: phospholipids 1: 25 w/w; 1: 4,208 mol/mol ratio). As shown in Fig. 5, A and B, the inhibitors tariquidar, elacridar and zosuquidar at 10 μM concentration still stimulated the ATP hydrolysis mediated by both m and hP- gp. These observations suggest that due to the use of high concentrations of DDM (40 mM or 333 X CMC during solubilization and
1.35 mM or 11.25 X CMC during purification), addition of phospholipids did not dilute the detergent sufficiently, or that the primary (high-affinity) binding site of P-gp was occupied by DDM detergent molecules and therefore addition of phospholipids could not displace DDM. Furthermore, when we increased the P-gp: phospholipid ratio to 1: 100 (wt. /wt.) or 1: 16,832 (mol/mol) in the ATPase assay, similar stimulation of ATPase activity was observed (data not shown) suggesting the latter possibility is most likely.Reconstitution of Purified mP-gp into Proteoliposomes Restores the Inhibitory Effect of Tariquidar and Elacridar on ATPase Activity—The above observations demonstrated that addition of excess phospholipids to the detergent micelles did not restore the ligand-P-gp interactions to those observed in native membrane environment.
A well-established method of replacing DDM is by forming proteoliposomes through slow removal of detergent in the presence of Bio-beads SM2 and phospholipids. We therefore reconstituted mP-gp into proteoliposomes using an E. coli acetone ether washed phospholipid mixture, as described in Experimental Procedures. The resulting proteoliposomes were collected by ultracentrifugation and used for ATPase assays, as detailed in Experimental Procedures. The vanadate-sensitive ATP hydrolysis of the reconstituted mP-gp was then evaluated in the presence of tariquidar and elacridar. As shown in Fig. 6, A and B, when the detergent was removed by bio- beads SM2 in mP-gp proteoliposomes, both tariquidar and elacridar inhibited ATP hydrolysis in a concentration-dependent manner similar to their observed effect in native membranes (Fig. 1). It should be noted that the IC50 values of these inhibitors for inhibition of ATP hydrolysis in proteoliposomes were comparable to those observed in native membranes (Table 1). We further evaluated the effect of tariquidar on the photolabeling of mP-gp with IAAP in proteoliposomes (Fig. 6C). As observed in native membranes, tariquidar at 1 µM in this case inhibited >90% of the photolabeling of mP-gp with IAAP, demonstrating that replacing DDM micelles with membrane lipid bilayer allows tariquidar access to its high- affinity binding pocket (Fig. 6C).
Removal of Detergent by Reconstitution of P-gp in Nanodiscs also Restores Inhibition of ATP Hydrolysis by Tariquidar and Elacridar— Membrane proteins including ABC transporters can also be reconstituted in a membrane-like environment using a combination of membrane scaffold protein (MSP) and lipids, thereby forming nanodiscs (22). In addition, nanodiscs offer an advantage of accessibility to both extracellular and intracellular regions of the transporter. The resultant nanodiscs thus keep membrane proteins in a defined native- like phospholipid bilayer environment with ligand access from both cytoplasmic and extracellular sides that provides stability to the protein compared to detergent micelles. In view of the above advantages of nanodiscs over proteoliposomes, mP-gp was reconstituted in nanodiscs as described in Experimental Procedures. Purified mP-gp in DDM micelles and nanodiscs reconstituted with purified protein was resolved on 7% Tris- Acetate gel and stained with InstantBlue. As shown in Fig. 7A lane 2, the nanodiscs fraction contains both mP-gp and MSP protein. The effect of tariquidar and elacridar on the ATP hydrolysis of mP-gp reconstituted in nanodiscs was then evaluated. As expected, contrary to their effect on mP-gp in detergent micelles, tariquidar and elacridar inhibited the ATP hydrolysis of mP-gp in nanodiscs (Fig. 7 B and C). However, the maximum inhibition was observed to be only 60-65% and the IC50 values of 1.55 ± 0.38 and 1.18 ± 0.22 µM for tariquidar and elacridar were significantly higher compared to those observed with proteoliposomes (Fig. 6), indicating that the affinity for these drugs is lower in nanodiscs. Because we used the same E. coli acetone-ether washed phospholipid mixture for reconstitution of purified P-gp in nanodiscs and proteoliposomes, the difference is not due to the lipid. Although it is likely that the MSP belt protein influences the access of tariquidar to its binding site, we do not have any definitive explanation at present for the observed differences. However, it is very clear that the inhibitory effect of tariquidar and elacridar on ATPase activity is recovered in nanodiscs.
Molecular Modeling Studies—Docking studies of tariquidar and DDM at the substrate-binding pocket of P-gp were carried out to find an explanation at the molecular level for the remarkable change in the biochemical behavior of P-gp in lipid membranes versus detergent micelles. As previously reported, the program AutoDock Vina finds many high score poses for tariquidar in the substrate-binding pocket of hP-gp using a homology model (4). The 10 poses with the best scores for hP-gp are shown in Fig. 8A. The same docking analysis of tariquidar was carried out for mP-gp using the 3.4 Å resolution X-ray structure 4Q9H.pdb (9); the poses with the highest scores are shown in Fig. 8B.
Docking of DDM was also carried out in hP- gp and mP-gp, using the same models and program settings. The detergent poses with the highest scores are shown in Fig. 8 on the right for both models, along with the docking of tariquidar and scores on the left. These modeling studies suggest that both ligands, tariquidar and DDM, bind to P-gp at the same site, as shown when tariquidar and DDM poses are presented in the same figure (compare positions of tariquidar and DDM in panels A and B). The docking scores of DDM (-7 to -9 kcal/mol) were numerically higher than the scores of tariquidar (-11 to -13 kcal/mol), reflecting the fact that tariquidar binds P-gp with higher affinity than DDM. It is very likely that due to a high concentration of detergent used to solubilize the membranes and to maintain the purified protein in soluble form, DDM occupies the binding site and cannot be displaced by tariquidar.One of the poses shown in Fig. 8A fully matches the mutagenesis and biochemical data of the interaction of tariquidar with hP- gp, as studied in isolated native membranes (4). This pose of tariquidar shown in Fig. 9, panel A (native membranes) represents the site where tariquidar binds and inhibits the basal ATP hydrolysis of hP-gp. As schematically represented in Fig. 9B, tariquidar thus binds to a suggested secondary or alternative low affinity site (compare IC50 and EC50 values in Table 1), where it produces stimulation of the basal ATP hydrolysis of P-gp. The residues lining the secondary low affinity site are not known at present. The presence of DDM surrounding the hydrophobic region including the drug- binding pocket of hP-gp is supported by cryo- EM studies with hP-gp (13), where DDM micelles are clearly shown to surround the transmembrane region of P-gp, as depicted in Fig. 9B.
Discussion
ABC transporters represent one of the largest transport protein families, with diverse physiological functions that are clinically important for certain disease conditions. Therefore, understanding the structure and function of these transporters in their native environment is critical for advancing our understanding of their physiological and pathological roles. The approach adopted to study the structure of the proteins has always been to crystallize the proteins after purification. Eukaryotic ABC transporters are normally expressed in heterologous systems including Pichia and baculovirus-infected insect cells and purified using a high concentration of detergent, significantly above the critical micelle concentration (CMC), the concentration above which micelles are formed spontaneously, to maintain the protein in active conformations. These conditions are optimal to obtain structural information, but do not allow the protein to remain in its native membrane-like environment.As both hP-gp and mP-gp recognize and transport a variety of chemically dissimilar amphipathic or hydrophobic compounds including toxins and anticancer agents (2,23,24), and a number of detergents have been reported to interact as ligands or “allocrits” with this multidrug transporter when tested in native membranes (21,25,26), we investigated the interactions of selected modulators and substrates with purified mP- gp and hP-gp in the presence of DDM micelles. In addition, we compared the differences in the activity of these transporters when they are present in native or artificial membranes and in detergent micelles.
We demonstrated that altering the membrane environment of P-gp has profound effects on its biochemical properties. While tariquidar, elacridar and zosuquidar inhibit P- gp-mediated ATP hydrolysis when the protein is present in native membranes, the same inhibitors actually stimulate the ATP hydrolysis mediated by purified protein in detergent micelles (Fig. 1). This change in interaction with the ligands was also demonstrated by tariquidar’s significantly decreased ability (at least 100-fold lower compared to native membranes) to compete for photoaffinity labeling of purified mP-gp with IAAP (Fig. 3).Our observations with modulators and stimulators of ATPase activity verapamil and QZ59 SSS, which also exhibit significantly reduced affinity for mP-gp in detergent micelles (Fig. 2 and Table 1) clearly show that somehow a detergent environment alters the conformation of the substrate-binding pocket of the transporter, thereby altering the access of inhibitors or substrates to the pocket. One likely possibility is that the DDM molecules occupy the primary substrate-binding site in purified P-gp, thereby blocking access of tariquidar to its preferred binding site. Tariquidar then binds to a secondary site with altered (decreased) affinity resulting in a switch from inhibition to stimulation of ATPase activity of P-gp. A similar switch from inhibition to stimulation of hP-gp-ATPase activity in native membranes by tariquidar, elacridar and zosuquidar was recently observed by us when the three residues Y953, Q725 and Y307 were substituted with alanine in the substrate-binding pocket (4). We found that substitution of two tyrosine residues and one glutamate residue with alanine disrupts the high-affinity interaction of these three modulators with P-gp, which is similar to our observations reported here with purified wild-type P-gp in a DDM micelle environment. In addition, P-gp has been shown to interact with alkyl phospholipids, edelfosine, and ilmofosine (27), suggesting that lipids or detergents can occupy the substrate-binding site in P-gp. Direct proof of these observations was provided in a recent study that showed lipid and/or detergent molecules in what may possibly be the substrate-binding pocket of mP-gp using mass spectrometry analysis (28). Thus, lipid molecules could interact with the mP-gp in the substrate-binding pocket as well as at the interface between the protein and the lipid bilayer. Occupation of the substrate-binding pocket by lipids might also explain the basal ATPase activity observed for P-gp, as has been reported earlier (29). In addition, several point mutations in the substrate- binding pocket of P-gp were found to result in a significant decrease in the basal ATPase activity (30).
X-ray crystallographic studies have also shown that detergent molecules can bind in the central cavity of P-gp (7), in a similar way to what docking studies of DDM in hP-gp and mP-gp have indicated (Fig. 6). The X-ray structure of C. elegans P-gp revealed two molecules of the detergent undecyl 4-O-α-D- glucopyranosyl-1-thio-β-D-glucopyranoside, with their disaccharide head groups inside the central cavity of the protein (7). One of these detergent molecules is in close proximity to some of the DDM poses generated in our molecular modeling studies, thereby providing strong evidence that detergent molecules can occupy the substrate-binding pocket of P-gp. Additionally, the presence of two molecules of another detergent, nonyl- glucopyranoside was reported in the ligand- binding cavity of the antibacterial peptide transporter McjD (31) and DDM was found bound to the transmembrane helices and a portal between transmembrane helices 1 and
2 in the X-ray crystal structure of the mitochondrial ABCB10 transporter (32).Based on our experimental and docking results, we propose a model schematically shown in Fig. 9. When a high affinity ligand such as tariquidar binds to the substrate- binding pocket of P-gp in the native membrane environment, it inhibits ATP hydrolysis.
In the case of purified protein, the detergent, which is used at very high concentrations during membrane solubilization and purification, may occupy the high-affinity site in the substrate-binding pocket. Therefore, tariquidar cannot access its primary binding site in the substrate- binding pocket. Instead, it binds to an alternative site (referred to as a secondary site (30)) resulting in concentration- dependent stimulation of ATP hydrolysis. Such modulation of P-gp ATP hydrolysis by detergents in the absence of any ligand was studied earlier by Orlowski et al. (33). Their work showed that detergents present at concentrations above their CMC during purification had a significant effect on the ATP hydrolysis of P-gp. Their report, however, did not address the effect of ligands in the detergent micelle environment on the ATP hydrolysis of the protein. Our data show that the substrate-binding pocket of purified P-gp when present in DDM micelles does not represent a native conformation (Fig. 9B). The native conformation of this pocket is restored when P-gp is reconstituted into proteoliposomes with similar properties as in native membranes (Fig. 9 A), where the detergent molecules are replaced by lipids. This is demonstrated by restoration of tariquidar’s ability to inhibit the ATP hydrolysis by P-gp and inhibition of IAAP binding to the substrate-binding pocket of the transporter. We further demonstrated the importance of lipids in maintaining the native state of P-gp and its interaction with ligands by incorporating P-gp into nanodiscs. As shown in Fig. 7 B and C, tariquidar was able to inhibit the ATPase activity of the purified protein in nanodiscs, although with a lower affinity.
Clearly, studying ligand interactions using purified P-gp in DDM micelles and excess phospholipids might not provide information about high-affinity binding sites in the substrate-binding pocket of this transporter. Loo et al first reported stimulation of purified P-gp ATPase activity by tariquidar in the presence of DDM micelles and lipids (21), but this observation was not consistent with other published work using native membranes. They also mapped tariquidar’s binding sites (34) on P-gp, although these are most likely low affinity binding sites, as the ATPase activity of purified mutant proteins was measured in a buffer containing DDM micelles and lipids. Our findings thus demonstrate that it is critical to study ligand interactions with P-gp in the detergent-free environment using native or artificial membranes. Consistent with our results, significant differences in conformations and biophysical properties of the bacterial MsbA transporter in DDM micelles and nanodiscs were recently reported (35) .
In summary, our data demonstrate that the environment of the substrate-binding pocket of P-gp determines its interaction with various substrates and the preferred binding sites for ligands in the detergent micelle environment are not accessible. The structural information about the ligand binding sites obtained with protein in DDM micelles by X-ray crystallography alone should be interpreted with caution. Similarly, the recently proposed role of individual helical movements in the determination of the polyspecificity of mP-gp (6) will have to be evaluated again using purified protein reconstituted in artificial lipid membrane. The inaccessibility of high-affinity sites on purified P-gp for modulators and substrates in detergent micelles can also explain the failure to co-crystallize transporters with high- affinity modulators or substrates. We suggest that new methods to keep P-gp and other ABC drug transporters in a detergent-free environment (much closer to that found under physiological conditions) are required for correlation of structural, biochemical, and biophysical studies.Materials—Anagrade DDM and other detergents were obtained from Anatrace (Maumee, OH). E. coli acetone/ether washed phospholipids and bovine phospholipids were purchased from Avanti Polar Lipids, Inc., (Alabaster, Al) and Ni-NTA resin from (Qiagen Inc., Valencia, CA). [125I]-Iodoarylazido- prazosin (IAAP; 2200Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). Bio Beads-SM-2 were obtained from Bio-Rad Laboratories (Hercules, CA). All other chemicals were purchased from Sigma- Aldrich Co. (St. Louis, MO).
Expression of Human and Mouse P-gp in Baculovirus-Infected High-Five Insect Cells and Preparation of Membranes—High-Five insect cells (Thermo Fisher Scientific, Waltham, MA) were infected with baculovirus carrying wild- type hMDR1(36) or M107L mmdr1a (37) with a 6-histidine tag at the C-terminal end and total membranes from infected cells were prepared following hypotonic lysis, as described previously (15) with minor modification. Aliquots of membranes were quickly frozen in dry ice and stored at -80 ºC.
Purification of hP-gp and mP-gp—The protein was purified as described earlier (13). Briefly, the membranes (150–250 mg of protein) were solubilized by using 2% n- dodecyl β-D-maltoside (DDM) in solubilization buffer (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 15% glycerol, 5 mM β-mercaptoethanol, 20 mM imidazole, UltraCruz EDTA-free protease inhibitor cocktail tablets (Santa Cruz Biotechnology, Dallas, TX). The supernatant was separated from solubilized extract by centrifugation at 38,000 rpm (Ti-45 rotor; Beckman Coulter, Brea, CA) for 45 min at 4°C. The supernatant containing solubilized protein was incubated with Ni-NTA resin (Qiagen Inc., Valencia, CA) pre-equilibrated with column buffer [(50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 15% glycerol, 5 mM β- mercaptoethanol, 20 mM imidazole, 0.04% sodium cholate, 0.0675% n-dodecyl β-D- maltoside (DDM)] for 14–16 h at 4°C. The metal resin was washed 5X with column buffer and P-gp was eluted with the same buffer containing 300 mM imidazole. The eluted protein was further purified using size exclusion chromatography using Superdex 200 10/300 GL in gel filtration buffer (50 mM Tris-HCl pH 7.5, 2% glycerol for mP-gp or 15% for hP-gp, 200 mM NaCl, 5 mM β-ME, 0.0675% DDM, 0.04% sodium cholate). The fractions containing monomer P-gp were collected and concentrated using Amicon 100 Kda concentrators (Thermo Fisher Scientific, Waltham, MA). 10 mM DTT was added to the concentrated protein fraction before quick- freezing in dry ice and storage at -80°C.
Reconstitution of P-gp in Nanodiscs and Proteoliposomes—Purified hP-gp or mP-gp was reconstituted into nanodiscs using a recombinant membrane scaffold protein MSP1E3D1 and E. coli total phospholipids, as described earlier with minor modifications [(38)]. Briefly, purified hP-gp or mP-gp was mixed with purified MSP1E3D1 (a gift from Van Que and Andrew Stephen, Leidos Inc, Frederick, MD) and sonicated E. coli phospholipids in 20 mM sodium cholate in a molar ratio of 1:10:110. The mixture was then incubated at room temperature with constant agitation for 1 h. To initiate removal of DDM and assembly of nanodiscs, Bio-beads SM-2 (0.6 g/mL) were added and incubated at 4°C for 12-14 h with constant agitation. Reconstituted nanodiscs were separated from the Bio-beads with a 25-gauge needle and further purified with size exclusion chromatography using Superdex 200 10/300 GL in a gel filtration buffer. The peak fractions were collected, concentrated with 100 Kda cut off concentrators and stored at 4 °C until used. In addition, empty nanodiscs (without P-gp) were also prepared and employed as controls in protein estimation and ATP hydrolysis Zosuquidar assays.