Interaction of the P-Glycoprotein Multidrug Transporter with Sterols

Oct 20, 2015 - Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors...
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Interaction of the P‑Glycoprotein Multidrug Transporter with Sterols Adam T. Clay, Peihua Lu, and Frances J. Sharom* Department of Molecular and Cellular Biology and Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada N1G 2W1 S Supporting Information *

ABSTRACT: The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3−9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

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phospholipids through two different nonexclusive mechanisms: specific interaction of a lipid with a binding site or regulation arising from nonspecific lipid-induced changes in the membrane environment of the protein.15 Cholesterol also plays an important role in regulating the function, folding, and stability of integral proteins. As with phospholipids, two modes of action are possible: direct interaction of the sterol at specific protein sites and indirect effects on the biophysical properties of the membrane.16 Because cholesterol alters membrane packing, order, and fluidity, disentangling these two effects can often be difficult. High-resolution structures of several integral proteins have revealed cholesterol bound at specific surface sites (see ref 16). Pgp is known to operate as a hydrophobic “vacuum cleaner”, expelling its substrates directly from the membrane, so it is not surprising that its function is broadly modulated by membrane composition and properties (reviewed in ref 17). Drug binding, drug transport, and ATPase activity are all sensitive to the gel to liquid-crystalline phase transition in proteoliposomes,18−21 and the lateral packing density of the bilayer has been proposed to alter the thermodynamic parameters of ATP hydrolysis.22 The drug binding affinity of Pgp is sensitive to the nature of the

he ATP-binding cassette (ABC) superfamily in humans contains 48 members, which are typically involved in energy-dependent transport of substrates across membranes. ABC transporters typically comprise two membrane-embedded domains, each containing six transmembrane helices, and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Mutation or altered expression of these transporters can result in disease and drug resistance. The best-studied ABC protein, P-glycoprotein (Pgp, ABCB1), is a 180 kDa multidrug efflux pump1−3 that has been linked to the emergence of multidrug resistant (MDR) cancers4 and Alzheimer’s disease.5 In recent years, X-ray crystallographic studies have yielded the structures of a number of ABC proteins,6 including Pgp,7 in distinct conformational states, some bound to nucleotides and/ or their substrates. In the absence of substrates, Pgp has high levels of basal ATP activity uncoupled from transport, which proceeds by a mechanism alternating between the two NBDs.8−10 How ATP hydrolysis drives the unidirectional transport of substrate remains unclear, but the basal ATPase cycle and ATP-driven drug transport cycle of Pgp appear to have distinct transition state conformations.11 Phospholipids are known to modulate the structure and function of membrane transporters, and both X-ray crystallographic structures (e.g., refs 12 and 13) and functional studies (e.g., ref 14) have revealed the existence of specific lipidbinding sites. Integral protein function may be regulated by © XXXX American Chemical Society

Received: August 14, 2015 Revised: October 19, 2015

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DOI: 10.1021/acs.biochem.5b00904 Biochemistry XXXX, XXX, XXX−XXX

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phospholipid headgroup, acyl chain composition, and fluidity.19 Both basal and drug-stimulated ATPase activity are also modulated by lipid composition.23,24 Furthermore, some lipids are able to stabilize the ATPase activity of detergent-solubilized Pgp.24,25 Pgp also has direct functional links to membrane lipids; it interacts with platelet-activating factors and various lipid-based anticancer drugs,26 and the reconstituted protein is known to be a flippase for various fluorescent phospholipids and glycolipids.27,28 The structure, function, and stability of other ABC transporters in mammals, yeast, and bacteria can be similarly modulated by lipid composition (e.g., ABCG2,29 ABCA4,25 HorA,30 and the maltose transporter31). Cholesterol can also modulate the function and stability of Pgp. The ATPase activity of Pgp was reduced by the removal of cholesterol from membranes,32 or reconstitution of Pgp into membranes without cholesterol.33 In addition, cholesterol affects both basal and drug-stimulated ATPase activity.33−36 Cholesterol also modulates Pgp transport function via its effects on drug partitioning into phospholipid bilayers,36 the extent of which is greatly reduced by the sterol.36,37 The observations that Pgp co-eluted with [3H]cholesterol, and the sterol altered its drug interactions,38 led to the suggestion that cholesterol might interact directly with the transporter. The “cholesterol fill-in model” based on these studies suggests that the sterol may occupy empty space in the substrate-binding pocket.38,39 Reconstituted Pgp is highly active in bilayers of synthetic phospholipids alone (e.g., ref 21); therefore, there is no strict functional requirement for cholesterol, and Pgp is likely not a cholesterol “flippase”36 as reported previously.32 However, the transporter is clearly modulated by sterols, and the nature of its interactions with them remains enigmatic. Cholesterol is very challenging to use. It has very low true solubility; its critical micelle concentration (CMC) is in the range of 25−40 nM.40 At higher concentrations, it appears to be “soluble” but actually exists as micelles or larger aggregates. It has no useful spectroscopic or fluorescence properties, and only one polar group, -OH, so even small modifications to its structure can dramatically alter its behavior. Fluorescent sterols with higher water solubility have been widely used in the study of membrane protein−sterol interactions.41 Dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol (DHE)] and cholestatrienol [5,7,9(11)cholestriene-3β-ol (CTL)] are good cholesterol mimics in terms of their biophysical behavior and membrane interactions, because they differ from cholesterol only by the presence of additional double bonds. Protein−sterol interactions have also been investigated using various cholesterol derivatives labeled with extrinsic fluorophores (e.g., ref 42). The water-soluble cholesterol analogue, cholesterol hemisuccinate (CHS), mimics cholesterol well43 and has similar effects on lipid order when compared to those of cholesterol.44 CHS increases membrane protein activity and stability45−47 and has proven to be useful in probing sterol−membrane protein interactions. In this study, the interaction of purified Pgp with various fluorescent and nonfluorescent sterol derivatives, including CHS, was investigated in detergent solution and lipid bilayers, using several complementary experimental approaches.48 Results suggested a direct, intimate association between the protein and several of these sterols, perhaps via specific binding regions. CHS also affected the folding of Pgp, and the functional stability of its catalytic and drug transport cycles, suggesting that sterols may interact directly with Pgp to modulate its structure and function.

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MATERIALS AND METHODS

Materials. CHAPS was obtained from MP Biomedicals (Solon, OH). Hoechst 33342 (H33342) was purchased from Invitrogen (Burlington, ON). ATP, DHE, CHS, cholesterolSO4, cholesterol-polyethylene glycol 600 sebacate (cholesterolPEG), and N-acetyl-tryptophanamide (NATA) were obtained from Sigma-Aldrich Canada (Oakville, ON). CHS-Tris salt was prepared from CHS. Phospholipids were acquired from Avanti Polar Lipids (Alabaster, AL). Egg PC was 99% pure. 22-[N-(7Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen3-ol (NBD-cholesterol) was obtained from Molecular Probes (Eugene, OR). CTL was provided by F. Maxfield (Department of Biochemistry, Weill Cornell Medical College, New York, NY). Sterols were added to detergent-solubilized Pgp from aqueous stock solutions (CHS Tris salt and cholesterol-PEG), or stock solutions in ethanol (DHE, CTL, and NBDcholesterol). Pgp Purification and Reconstitution. Plasma membrane vesicles were isolated from MDR CHRB30 cells as previously described.49 Pgp was purified from plasma membrane using a modified version of an established protocol,36 by initial extraction with 15 mM CHAPS buffer in HEPES buffer [20 mM HEPES, 100 mM NaCl, and 5 mM MgCl2 (pH 7.4)], followed by solubilization of the pellet in 45 mM CHAPS/ HEPES buffer to produce partially purified Pgp. This was further purified by affinity chromatography using two passes through a concanavalin A-Sepharose 4B column pre-equilibrated with 2 mM CHAPS/HEPES buffer. Purified Pgp (90− 95% pure49) had high ATPase activity (∼2 μmol min−1 mg−1) and a protein concentration of >350 μg mL−1. Reconstitution of Pgp was conducted using gel filtration chromatography as previously described,27 with the following modifications. For proteoliposomes containing only phospholipids, 3 mg of stock lipid in 4:1 (v/v) CHCl3/MeOH solvent was dried under nitrogen and then pumped in a vacuum for 30 min. For samples containing CHS, 2.5 mg of a phospholipid stock solution and 0.5 mg of CHS in 4:1 (v/v) CHCl3/MeOH solvent were mixed in the same tube before being dried. Dried lipid was solubilized in 75 μL of 500 mM CHAPS/HEPES buffer and mixed with 1 mL of purified Pgp containing 250−300 μg of protein. CHAPS was removed when the mixture was passed through a Sephadex G50 column (1 cm × 27 cm), and the fractions containing proteoliposomes were identified by turbidity and ATPase activity. A CHAPS assay50 showed that the proteoliposomes did not contain detectable levels of detergent. Protein concentrations were determined using the method of Bradford.51 Sterol Binding Affinity. The affinity of Pgp for binding DHE and NBD-cholesterol was determined by quenching titration of the intrinsic Trp fluorescence of detergentsolubilized Pgp, in the absence of added exogenous phospholipids.36,52 Fluorescence emission was monitored at 22 °C using a PTI QuantaMaster C-61 or QM-8/2005 steady state fluorimeter (Photon Technology International, Edison, NJ) with excitation and emission at 290 nm (2 nm slits) and 330 nm (4 nm slits), respectively. Curves were baselinecorrected for nonspecific quenching and inner filter effects by performing an identical titration using the soluble Trp analogue NATA (using excitation at 290 nm and emission at 330 nm), and the fluorescence intensities of Pgp and NATA were normalized. Quenching of Trp fluorescence at each sterol B

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Figure 1. Chemical structures of the sterols used in this study: dehydroergosterol (DHE), cholestatrienol (CTL), 22-[N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol), and cholesterol hemisuccinate (CHS).

version of a real-time fluorescence-based assay.26 A 100 μL aliquot of Pgp proteoliposomes containing 5 μM H33342 was mixed with 100 μL of HEPES buffer and incubated at 37 °C for 10 min. The diluted proteoliposome sample was then added to a 0.5 cm × 0.5 cm quartz cuvette at 37 °C. H33342 was excited at 355 nm (2 nm slits), and its emission was monitored continuously at 450 nm (2 nm slits) for 100 s. At this time, 50 μL of temperature-equilibrated ATP together with a regenerating system (25 mM ATP, 11.5 mg/mL creatine phosphate, and 0.3 mg/mL creatine kinase in HEPES buffer) was added to initiate transport, and the fluorescence was monitored at 450 nm. Less than 2% of the total fluorescence emission signal arose from aqueous drug (data not shown); therefore, it was assumed that lipid-bound drug was responsible for all observed fluorescence emission. The initial drug transport rate (percent fluorescence change per minute) was determined by linear regression from the slope of the plot of the percent control fluorescence emission intensity (relative to 100% at the time of ATP addition) versus time, from 5 to 15 s following ATP addition. The initial concentration of drug in the membrane depends on the phospholipid composition of proteoliposomes,19 and therefore, the initial fluorescence intensity and initial rate obtained using this method cannot be directly compared for proteoliposomes with differing compositions. Thus, percent control values relative to the transport activity at a reference time point were used for the purpose of comparison. Stability of ATPase and Transport Activity. NaN3 [0.02% (w/v)] was added to all buffers to prevent microbial growth over the extended incubation period. CHS-Tris salt was used to test the effect of CHS on detergent-solubilized Pgp because it is more water-soluble than the free acid form. CHSTris salt, DHE, cholesterol-SO4, and NBD-cholesterol were added to purified Pgp at the indicated concentrations. ATPase assays (in the absence of added exogenous phospholipids)53 and transport assays (as described above) were performed at 24 h time intervals. To measure H33342-stimulated ATPase

concentration was fitted to the following equation using SigmaPlot (Systat, San Jose, CA) ΔF × 100 = F0

(

ΔFmax Fo

)

× 100 [S]

Kd + [S]

(1)

where (ΔF/F0) × 100 represents the percent change in fluorescence intensity relative to the initial value after addition of sterol at concentration [S] and (ΔFmax/Fo) × 100 is the maximal percent fluorescence quenching. Interaction of CTL with detergent-solubilized Pgp in the absence of added exogenous phospholipids was conducted using a method involving incubation of individual Pgp samples with different CTL concentrations at 4 °C for 2 h, as described previously for the sterol-binding protein NPC1.42 CTL quenching of Pgp intrinsic Trp fluorescence was monitored using excitation at 290 nm and emission at 330 nm (see above), and enhanced fluorescence of CTL was monitored using excitation at 325 nm and emission at 375 nm. Collisional Quenching of NBD-Cholesterol. Freshly prepared stock solutions of 5 M KI in 15 mM CHAPS/ HEPES buffer were added as 1 μL aliquots to 100 μL of 0.2 or 10 μM NBD-cholesterol in the presence or absence of 70 μg/ mL Pgp in 15 mM CHAPS buffer. To prevent I3− formation, 0.1 mM Na2S2O3 was added to the KI stock solution. NBDcholesterol was excited at 470 nm, and the emission intensity was monitored at 540 nm. Fluorescence intensities were corrected for dilution, and scattering was corrected by titration of buffer alone with KI. Determination of the CMC of NBD-Cholesterol. The CMC of NBD-cholesterol was estimated by collecting fluorescence polarization data for increasing concentrations of the fluorescent lipid in HEPES/15 mM CHAPS buffer using an L-format, and calculations were performed using the instrument software (Felix32). Initial Rates of Drug Transport. Initial rates of Pgpmediated H33342 transport were determined using a modified C

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Figure 2. Quenching of the intrinsic Trp fluorescence of purified Pgp at 22 °C by fluorescent sterol derivatives. (A) Trp quenching titration curve for the interaction of CHAPS-solubilized Pgp with DHE. (B) Trp titration curve for the interaction of CHAPS-solubilized Pgp with NBD-cholesterol. (C) Trp titration curve for the interaction of Pgp reconstituted into egg PC proteoliposomes with NBD-cholesterol. Trp fluorescence was monitored using excitation at 290 nm and emission at 330 nm. (D) Fluorescence polarization titration of NBD-cholesterol in 2 mM CHAPS buffer. NBDcholesterol was excited at 470 nm, and emission intensity was monitored at 540 nm. The vertical line represents the CMC of NBD-cholesterol, as defined by the intercept of the two linear regression lines in the low and high concentration ranges.

activity, 5 μM H33342 was added before initiation of the reaction with ATP, to match the final conditions of the transport assay. A paired t test or signed ranked test (where appropriate) was used to determine whether the stability of basal ATPase activity, H33342-stimulated ATPase activity, and H33342 transport was significantly increased by CHS (p < 0.05) over the 7 day period. Circular Dichroism Spectroscopy. The thermal denaturation and far-UV CD spectrum of CHAPS-solubilized Pgp (70 μg/mL) were monitored using a Jasco J-815 CD spectrometer with a 2 mm path-length quartz cuvette. For protein denaturation experiments, the loss of negative ellipticity at 222 nm was monitored as the sample temperature was ramped from 25 to 90 °C in a Peltier-controlled cell, at a rate of 1.5 °C/ min, and data were collected with a digital integration time of 4 s. HT voltage data were collected concomitantly. CD spectra were obtained at 22 °C with a digital integration time of 1 s and a scan range of 190−250 nm. CHS (Tris salt) and cholesterolPEG were added to Pgp from aqueous stock solutions, whereas DHE and NBD-cholesterol were added from ethanol stock solutions [4% (v/v) final concentration]. The corresponding Pgp controls were treated with either aqueous buffer or 4% (v/ v) ethanol, which had no effect on Pgp ATPase activity. Identification of Putative Cholesterol-Binding Sites in Mouse Pgp. The Expasy Bioinformatics Tool, ScanProSite, was used to search for CRAC motifs [(L/V)-X1−5-(Y)-X1−5-(K/ R)] and CARC motifs [(K/R)-X1−5-(Y/F)-X1−5-(L/V)] in the mouse Pgp sequence, which were mapped onto the X-ray crystal structure of mouse Pgp (Protein Data Bank entry

3G5U) using PyMol. Motifs found within the transmembrane domains (TMDs) that had the correct membrane orientation were identified as putative cholesterol-binding sites.54 Membrane orientation was estimated using the bilayer location shown in Figure S1. The precise location of the lipid bilayer is not known and may depend on bilayer composition.

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RESULTS Interaction of Pgp with Fluorescent Sterols. The fluorescence properties of DHE, CTL, and NBD-cholesterol (Figure 1) allowed the demonstration of their direct interaction with Pgp. Titration of CHAPS-solubilized Pgp (2 mM, below the CMC; no added exogenous phospholipids) with increasing concentrations of DHE showed monophasic, saturable quenching of the protein Trp fluorescence, which was fitted to an equation describing binding of DHE to a single site with a dissociation constant of ∼4 μM (Figure 2A). However, titration of Pgp with increasing concentrations of NBD-cholesterol showed a biphasic binding curve, which did not fit a simple one-site model (Figure 2B). The discontinuity in the titration curve of Pgp with NBD-cholesterol occurred in the ∼100 nM range. A monomer-to-micelle transition could result in apparent two-phase binding, because the transition would alter the concentration of free monomeric NBD-cholesterol available to bind Pgp. Because micelles are larger than monomers, a decrease in fluorescence polarization would be expected at the critical micelle concentration. Indeed, a discontinuity was observed for the fluorescence polarization of NBD-cholesterol alone (Figure 2D), which is indicative of D

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accessible to the aqueous solution when it interacts with Pgp, dynamic quenching studies were conducted. A linear Stern− Volmer plot (Figure 4A) was observed for KI quenching of 0.2 μM NBD-cholesterol in 15 mM CHAPS (above the CMC), where the sterol is expected to be present within the detergent

the monomer-to-micelle transition, at a concentration similar to that seen in the titration with Pgp. Thus, the steep initial phase of quenching observed for Pgp in the presence of NBDcholesterol up to 100 nM arises from monomeric sterol, and the shallower second quenching phase above 100 nM arises from micellar sterol (Figure 2B). Titration of reconstituted Pgp in egg PC proteoliposomes with NBD-cholesterol resulted in a monophasic binding curve that yielded a Kd value of ∼4 μM (Figure 2C). In this case, the bilayer acts as a “sink” for NBDcholesterol, thus avoiding issues with a monomer-to-micelle transition. Thus, quenching of Pgp Trp residues by DHE and NBD-cholesterol suggests that the two sterols interact with the transporter in both detergent solution and lipid bilayers, with affinities in the low micromolar range. CTL also quenched the Trp fluorescence of CHAPS-solubilized Pgp in a saturable fashion (Figure 3A), yielding a Kd value of ∼6 μM. The

Figure 3. Quenching of the intrinsic Trp fluorescence of CHAPSsolubilized Pgp at 22 °C by increasing concentrations of CTL (A) and concomitant enhancement of the fluorescence emission of CTL (B). Trp fluorescence was monitored using excitation at 290 nm and emission at 330 nm; enhanced fluorescence of CTL was monitored using excitation at 325 nm and emission at 375 nm.

Figure 4. (A) Stern−Volmer plots for KI quenching at 22 °C of NBDcholesterol in CHAPS micelles (15 mM CHAPS buffer) in the absence and presence of purified Pgp. NBD-cholesterol was excited at 470 nm, and the emission intensity was monitored at 540 nm. (B) Fluorescence emission spectra of NBD-cholesterol in 15 mM CHAPS micelles at 22 °C in the absence and presence of purified Pgp; NBD-cholesterol was excited at 470 nm. (C) FRET between Pgp Trp residues and bound NBD-cholesterol at 22 °C. Trp fluorescence spectra of purified Pgp (50 μg/mL) in 2 mM CHAPS buffer in the absence () and presence (−−−) of 3 μM NBD-cholesterol, excited at 290 nm. The baseline fluorescences of CHAPS buffer alone (···) and CHAPS buffer containing 3 μM NBD-cholesterol (−·−) are also shown. The inset shows the enhanced fluorescence emission of NBD-cholesterol under the same conditions (excitation at 290 nm) in the presence of CHAPS-solubilized Pgp (−−−) compared to the fluorescence of Pgp alone (), and the baseline fluorescence of CHAPS buffer with (···) and without (−·−) 3 μM NBD-cholesterol.

fluorescence emission of CTL, which is very low in aqueous solution, was greatly enhanced on binding to Pgp (Figure 3B). These results suggest that CTL moves into a nonpolar environment when it interacts with Pgp. Fitting of the increase in CTL fluorescence emission intensity to an equation for a single binding site gave a Kd value (9 μM) similar to that observed for its quenching of Pgp Trp fluorescence. Nature of the Interaction between Pgp and NBDCholesterol. NBD-cholesterol could be associated with Pgp simply by being present in the same detergent micelle, or it could interact specifically with a protected binding site within the protein. To determine whether NBD-cholesterol is E

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Biochemistry micelles and fully accessible to the quencher. However, the addition of Pgp to NBD-cholesterol in CHAPS micelles resulted in a curved Stern−Volmer plot, which indicated that the Pgp-bound sterol was protected from quenching by KI. A blue shift in the fluorescence spectra of NBD-cholesterol was also observed when it was present together with Pgp in CHAPS micelles, when compared to those of CHAPS micelles alone (Figure 4B), which shows that the local environment of the fluorescent sterol is substantially less polar when it interacts with the transporter. Close interaction between the protein and NBD-cholesterol was also suggested by the observation of FRET between Pgp Trp residues and bound NBD-cholesterol (Figure 4C). The Trp emission intensity of CHAPS-solubilized Pgp was reduced in the presence of 3 μM NBD-cholesterol, and concomitant enhanced fluorescence was observed for NBDcholesterol compared to the sterol in CHAPS buffer alone. Taken together, these results suggest that NBD-cholesterol interacts intimately with Pgp at a protected site, rather than simply occupying the same detergent micelle. Effect of CHS on the Secondary Structure and Thermal Denaturation of Pgp. The CD spectrum of Pgp in 2 mM CHAPS buffer is indicative of a protein with a high αhelix content, displaying peak maxima/minima at 195, 210, and 222 nm (Figure 5A). The addition of CHS caused a decrease in the magnitude of the molar ellipticity and absorption (absorption data not shown), with little change in the peak maxima/minima (Figure 5A). The thermal denaturation of Pgp in 2 mM CHAPS was measured by monitoring the protein molar ellipticity as the temperature was increased (Figure 5B,D). A sigmoidal denaturation curve was observed for Pgp in the absence of sterols, with a melting point of ∼54 °C. The corresponding HT voltage curve did not show a large change around this temperature (Figure 5C), and there was no visible turbidity in the solution following completion of the thermal scan, which suggested that this transition did not result from protein aggregation.55 The normalized ellipticity reached around −0.5 at 80 °C, suggesting the existence of residual secondary structure, and the CD spectrum confirmed this (data not shown). The addition of CHS to Pgp resulted in a much broader transition, reduced the level of denaturation at all temperatures, and prevented the loss of ellipticity at 222 nm at temperatures up to 80 °C (Figure 5B). In contrast, cholesterolPEG, DHE, and NBD-cholesterol did not induce changes in the thermal denaturation profile of Pgp (Figure 5B,D). Ellipticity increased sharply for Pgp with cholesterol-PEG starting at temperatures close to 80 °C, which corresponded to a large increase in voltage, suggesting that protein aggregation took place at this temperature. Effect of Sterols on the Basal ATPase Activity of Detergent-Solubilized Pgp. Table 1 shows the initial ATPase activities of CHAPS-solubilized Pgp (with no added exogenous phospholipids) in the presence and absence of various sterols, which ranged from 1.1 to 2.4 μmol min−1 (mg of protein)−1. Cholesterol-PEG reduced the initial ATPase activity by ∼50%; this was the only sterol to cause a statistically significant difference in initial Pgp activity. The basal ATPase activity of Pgp in 2 mM CHAPS buffer declined by 50% after 48 h at 4 °C and reached 5-fold) when CHS was included in the bilayer. Egg PC proteoliposomes also produced low basal activity, but this was poorly stimulated by CHS. Asolectin proteoliposomes showed substantially higher basal ATPase activity, which was stimulated 2-fold by the presence of CHS to produce the highest catalytic activity of the six lipid combinations tested. Figure 7 shows that the stability of Pgp’s catalytic and transport cycles in proteoliposomes was also lipid-dependent. In addition, results indicated that while the stability of basal and drug-stimulated ATPase activity was often similar in different lipid environments, the transport activity appeared to be regulated independently and could display stability substantially lower than that of the catalytic activity. DMPC maintained the stability of basal ATPase activity better than a detergent solution (compare Figure 7A with Figure 6A), and this was substantially enhanced by the presence of CHS in the proteoliposomes, with ∼70% activity remaining after 7 days (Figure 7B). The stability of drug-stimulated catalytic activity and drug transport was significantly enhanced by DMPC relative to detergent solution, and addition of CHS further increased the stability of drug-stimulated ATPase activity, but not that of drug transport (Figure 7A,B). Essentially no change in ATPase stability was observed for Pgp reconstituted into egg PC relative to detergent solution, with or without CHS, with both activities dropping to ∼11% of the initial value after 7 days (Figure 7C,D). Similarly, Pgp transport activity was very poorly maintained in egg PC proteoliposomes, and CHS did not significantly increase its stability. Reconstitution into asolectin resulted in stabilization of basal and drug-stimulated ATPase activity, which was further increased by CHS (Figure 7E,F). Drug transport was also greatly stabilized by the presence of CHS in the asolectin bilayer. Importantly, the stability of the catalytic and transport cycles could be different in the same proteoliposome system. For example, in DMPC proteoliposomes, the stability of the transport cycle and drug-stimulated catalytic cycle was higher

Figure 6. (A) Stability of Pgp ATPase activity at 4 °C in CHAPS buffer (○) and in the presence of CHS (●; Tris salt, 0.32 mM) and cholesterol-PEG (◆; 0.32 mM). (B) Stability of Pgp ATPase activity in CHAPS buffer with 4% (v/v) ethanol (○), and in the presence of cholesterol-SO4 (◇; 0.32 mM), DHE (◆; 10 μM), and NBDcholesterol (●; 10 μM), which were all added from ethanol stock solutions [final concentration of 4% (v/v)]. Data are reported as the mean ± the standard deviation obtained from several independent experiments (n = 2−7).

for detergent-solubilized Pgp with CHS, with cholesterol-PEG, and without treatment, respectively (Figure 6A). The addition of 4% (v/v) ethanol to Pgp also greatly stabilized the ATPase activity, with no decrease observed after 7 days (Figure 6B). DHE had little effect on Pgp ATPase stability relative to the ethanol control, whereas cholesterol-SO 4 had a small destabilizing effect (Figure 6B). In contrast, the stability of Pgp ATPase was greatly reduced by NBD-cholesterol, with 95% C16+C18), where mismatch is less likely to be a problem. Because egg PC shows low basal activity with little CHS stimulation, while asolectin shows high basal activity with moderate CHS stimulation, factors other than hydrophobic mismatch are likely at play for these two lipids. In the case of egg PC, no stabilization of the catalytic/transport cycles by CHS was observed, and for asolectin, CHS stabilized all three cycles, again pointing to issues other than hydrophobic mismatch. When Pgp was reconstituted into proteoliposomes of three different phospholipids, the catalytic cycles (basal and drugstimulated) and the transport cycle behaved differently, in the absence and presence of CHS. While the basal and drugstimulated cycles often showed similar stability, the transport activity appeared to be regulated in a manner independent of the catalytic activity and was often substantially less stable. This suggests that the observed drug-stimulated ATPase activity may not be coupled to drug transport. Distinct cycles would have unique transition state conformations, as proposed previously.11 It was previously noted that synonymous variants of Pgp showed differing transport properties and responses to inhibitors, while their basal and drug-stimulated ATPase activities remained unchanged.62 Because direct measurement of substrate transport is difficult (and has not been achieved for most ABC proteins), substrate-stimulated ATPase activity is often used as a surrogate measure of transport activity. It is clear from this work that the drug-stimulated ATPase activity I

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Biochemistry

environment.71 Associations of Pgp with specialized membrane domains may represent a mechanism for functional regulation of the transporter in some cell types.17

and transport activity of Pgp are not always correlated, and conclusions about drug transport drawn from ATPase activity measurements should be viewed with caution. There are several possibilities for the location of the site of interaction of sterol with Pgp. A mass spectrometry study that examined association of phospholipids with Pgp suggested that lipid molecules might dock within the drug-binding cavity of the transporter.57 Although interaction with sterols was not examined, it was noted that bulky cardiolipin molecules also bound well to Pgp with a fixed stoichiometry. On the basis of this observation, sterols may indeed interact with Pgp by occupying the drug-binding cavity, as suggested by Ueda and co-workers.38,39,63 A recent X-ray crystallography study showed another independent binding site for drugs on the surface of mouse Pgp, facing the inner leaflet of the lipid bilayer.64 It is possible that this could be a sterol-binding site, although it might not protect bound sterol from quenchers, as we observed for NBD-cholesterol. Specialized CRAC/CARC motifs have been demonstrated to bind cholesterol in a number of integral membrane proteins,54 including the ABC transporter ABCG2.65 These motifs interact with several different moieties of the cholesterol molecule via van der Waals interactions with the iso-octyl chain and πstacking with the sterane rings.54 H-Bonding with the sterol -OH group may also occur but is not mandatory, and the Tyr group of the CRAC motif may be replaced by Phe in the CARC motif. In addition, the CRAC motif, both as a synthetic peptide fusion protein and in a form of HIV-1 gp160, has been reported to interact specifically with CHS-agarose (CHS lacks the -OH group).66 We identified four CARC motifs in the mouse Pgp sequence and located them at different bilayer depths within the TMDs in the three-dimensional X-ray crystal structure of the protein (Table S1 and Figure S1). One of the motifs is found at the gate to the substrate-binding pocket and could act to control substrate access. It is conceivable that some of these putative binding sites may interact with cholesterol. Which sites are able to do so could depend on where the sterol is located in a bilayer composed of specific phospholipids. This could explain why sterols can modulate the transport, drugstimulated, and basal ATPase activity differently depending on membrane composition, as observed in this study. Additional experimentation will be required to verify whether any of these motifs are indeed involved in binding cholesterol. It seems evident that cholesterol may regulate Pgp function by both direct and indirect mechanisms. This study has demonstrated that certain sterols interact intimately with the protein with affinities in the low micromolar range. Sterols may act as allosteric regulators of Pgp function, especially in the case of CHS, which appear to stabilize the activity, secondary structure, and folding of Pgp. Other membrane proteins have been reported to be affected by CHS in similar ways, including the human β2-adrenergic receptor,45 the human adenosine A2a receptor,67 and the neurotensin receptor.47 Indirect mechanisms of Pgp modulation by sterols include effects on substrate partitioning, and other membrane biophysical properties, such as fluidity and order.19,36,37 Cholesterol may also affect the lateral distribution of the transporter between membrane domains. It has been proposed that Pgp exists in two populations, one within and one outside of detergent resistant domains, which respond differently to cholesterol.68 There have been other reports that Pgp localizes to specific membrane domains,69,70 including cholesterol-rich lipid rafts and caveolae, and that it is functional in this type of

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b00904. Table S1 showing the location and characteristics of the CARC sequences identified as putative cholesterolbinding sites in mouse Pgp (PDF) Figure S1 showing the location and characteristics of the CARC sequences identified as putative cholesterolbinding sites in mouse Pgp (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (519) 824-4120, ext. 53362. Fax: (519) 837-1802. Funding

This research was funded by the Canadian Cancer Society (Grant 700248). We also thank the Canada Research Chairs program for their financial support of the research conducted in the authors’ laboratory. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Joseph Chu for large-scale growth of CHRB30 cells and preparation of the plasma membrane fraction. ABBREVIATIONS ABC, ATP-binding cassette; cholesterol-PEG, cholesterolpolyethylene glycol 600 sebacate; CHS, cholesterol hemisuccinate; CMC, critical micelle concentration; CRAC/CARC, cholesterol recognition/interaction amino acid consensus sequence; CTL, cholestatrienol; DHE, dehydroergosterol; DMPC, 1,2-dimyristoylphosphatidylcholine; H33342, Hoechst 33342; LDS-751, 4-[(1E,3E)-4-(3-ethyl-1,3-benzothiazol-3ium-2-yl)buta-1,3-dienyl]-N,N-dimethylaniline perchlorate; MDR, multidrug resistance/resistant; NATA, N-acetyl-tryptophanamide; NBD, nucleotide-binding domain; NBD-cholesterol, 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol; Pgp, P-glycoprotein; TMD, transmembrane domain.

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