1521-0111/88/1/84–94$25.00 http://dx.doi.org/10.1124/mol.114.096792MOLECULAR PHARMACOLOGY Mol Pharmacol 88:84–94, July 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

ATP–Binding Cassette Transporter Structure Changes Detectedby Intramolecular Fluorescence Energy Transfer forHigh-Throughput Screening s

Surtaj H. Iram1, Simon J. Gruber, Olga N. Raguimova, David D. Thomas,and Seth L. RobiaDepartment of Cell and Molecular Physiology (S.H.I., O.N.R., S.L.R.), Cardiovascular Research Institute (O.N.R., S.L.R.), StritchSchool of Medicine, Loyola University Chicago, Maywood, Illinois; and Department of Biochemistry, Molecular Biology, andBiophysics, University of Minnesota, Minneapolis, Minnesota (S.J.G., D.D.T); and Department of Chemistry and Biochemistry,South Dakota State University, Brookings, South Dakota (S.H.I.)

Received November 20, 2014; accepted April 29, 2015

ABSTRACTMultidrug resistance protein 1 (MRP1) actively transportsa wide variety of drugs out of cells. To quantify MRP1 structuraldynamics, we engineered a “two-color MRP1” construct byfusing green fluorescent protein (GFP) and TagRFP to MRP1nucleotide–binding domains NBD1 and NBD2, respectively.The recombinant MRP1 protein expressed and traffickednormally to the plasma membrane. Two-color MRP1 transportactivity was normal, as shown by vesicular transport of[3H]17b-estradiol-17-b-(D-glucuronide) and doxorubicin efflux inAAV-293 cells. We quantified fluorescence resonance energytransfer (FRET) from GFP to TagRFP as an index of NBDconformational changes. Our results show that ATP binding

induces a large-amplitude conformational change that bringsthe NBDs into closer proximity. FRET was further increasedby substrate in the presence of ATP but not by substratealone. The data suggest that substrate binding is required toachieve a fully closed and compact structure. ATP analogsbind MRP1 with reduced apparent affinity, inducing a partiallyclosed conformation. The results demonstrate the utility of thetwo-color MRP1 construct for investigating ATP-bindingcassette transporter structural dynamics, and it holds greatpromise for high-throughput screening of chemical librariesfor unknown activators, inhibitors, or transportable substratesof MRP1.

IntroductionThe ATP–binding cassette (ABC) proteins comprise one of

the largest superfamilies of membrane proteins and areubiquitous in all forms of life. The ABC transporters can beclassified as importers (bringing substrates into the cell) orexporters (expelling substrates from the cell). Both importersand exporters are present in prokaryotes, whereas onlyexporters are found in eukaryotes. The importance of ABCtransporters is highlighted by the fact that mutations inseveral members of the family are associated with humandisease (Sheppard and Welsh, 1999; Gottesman, 2002;Aittoniemi et al., 2009). ABC proteins are capable of trans-porting a wide variety of xenobiotics, including organic anions

from endogenous and exogenous sources, many of which areconjugated to glutathione [e.g., cysteinyl leukotriene, glucu-ronide (e.g., estradiol glucuronide, 17b-estradiol-17-b-(D-glu-curonide), E217bG), or sulfate (e.g., estrone sulfate, E2SO4)(Conseil et al., 2006; Deeley et al., 2006)]. This panspecificexport activity is relevant to clinical practice as it is the basisof physiologic resistance to a wide variety of drugs. Inparticular, overexpression of ABC transporters such asP-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1(MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) may lead to failure of chemotherapy in cancerpatients. These ABC transporters mediate ATP-dependentefflux of anticancer drugs across the plasma membrane,leading to reduced intracellular accumulation and conse-quently reduced efficacy of those drugs.The prototypical functional ABC transporter has a four-

domain core structure, composed of two membrane-spanningdomains (MSDs) and two cytoplasmic nucleotide-bindingdomains (NBDs) (Dean and Allikmets, 2001). Availablestructural data suggest that the transmembrane a-helices of

This work was supported by National Institutes of Health grants to S.L.R.[R01-HL106189, R01-HL092321] and D.D.T. [R01-GM27906], and by a giftfrom the McCormick Foundation to Loyola University Chicago.

1Current affiliation: Department of Chemistry and Biochemistry, SouthDakota State University, Brookings, South Dakota.

dx.doi.org/10.1124/mol.114.096792.s This article has supplemental material available at molpharm.aspetjournals.

org.

ABBREVIATIONS: ABC, ATP-binding cassette; AMP-PNP, adenylylimidodiphosphate; BMH, bismaleimidohexane; CV, coefficient of variance;DMSO, dimethyl sulfoxide; DOX, doxorubicin; E217bG, 17b-estradiol-17-b-(D-glucuronide); FRAP, fluorescence recovery after photobleaching;FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; MK571, 3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid; MSD, membrane-spanning domain; NBD, nucleotide-binding domain; NCC, NIHClinical Collection; PBS, phosphate-buffered saline; TIRF, total internal reflection fluorescence.

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the two MSDs are intertwined to form the substrate trans-location pathway and extend into the cytoplasm and makecontacts with the NBDs (Hollenstein et al., 2007). SeveralABC transporters, including MRP1, have an additional MSD(MSD0) at the amino-terminus of the protein. (Fig. 1A) (Hipfneret al., 1997). The precise function of MSD0 is not yet fullyunderstood. Structural studies of bacterial ABC transporterscaptured in a state reflecting ATP-bound conformation indicatethat the two cytoplasmic NBDs interact with each other ina head-to-tail orientation to generate two composite nucleotide-binding sites. The binding and hydrolysis of ATP provides theenergy required for the transport process (Hollenstein et al.,

2007). Despite significant progress in the field of ABC trans-porters, themolecular details of the transport process are still farfrom being fully understood.There is no high-resolution crystal structure available for

MRP1 or any of the other 12 subfamily members, but severalstructures have been reported for other ABC transporters.These studies have provided a wealth of information aboutstructure/function relationships, revealing the drug-bindingchamber, and providing insight into the molecular basis ofpolyspecificity. The data suggest an ABC efflux transportercan exist in at least two major conformations (Fig. 1B); an“inward conformation” and an “outward conformation.” The

Fig. 1. Expression, localization, andFRET of two-color MRP1. (A) Schematicdiagram of two-color MRP1 constructshowing an intrasequence GFP (twoamino acid linker on each side) and aC-terminus TagRFP (five amino acidlinker). (B) Crystal structure of mouseP-gp in apo condition showing the sepa-rated NBDs (left) (Aller et al., 2009);crystal structure of nucleotide-boundStaphylococcus aureus Sav1866 showingthe closed NBD structure (right) (Dawsonand Locher, 2006). (C) Immunoblots ofwhole-cell lysates (10 mg) containing wild-type (WT-MRP1) and two-color MRP1(2C-MRP1) compared with untransfectedcells (control), with detection by mousemonoclonal anti-MRP1 (1:5000 dilution,1 hour at room temperature) or anti-GFPantibody (1:2000 dilution, 1 hour at roomtemperature). (D) AAV-293 cells express-ing two-color MRP1 were plated on glass-bottom chambered coverslips as describedin experimental procedures. Fluorescentimages were collected using a Nikon in-verted microscope equipped with a 60�objective. Widefield lamp excitation andcomputer-controlled filter wheels wereused to acquire GFP and TagRFP images.(E) MRP1 membrane expression andlateral diffusion investigated by TIRFmicroscopy and FRAP of a 1.3-mm targetregion. (F) Data from FRAP were ana-lyzed using a nonlinear curve fit witha single exponential function. (G) MRP-1FRET did not depend on protein expres-sion level.

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inward conformation is the high substrate-affinity state, inwhich the binding pocket is facing toward the cytoplasm andthe two NBDs are separated. The outward conformation is thelow substrate affinity state with the binding pocket facingtoward the extracellular space and the two NBDs closelyinteracting. It is not clear whether the inward conformation(with widely open NBDs) is significantly populated in vivo.It is possible that physiologic concentrations of nucleotide(several millimolar) would maintain the NBDs in a closedconformation, and the amplitude of the conformational changemay be smaller than is suggested by the extreme states cap-tured by the crystal structures (Aller et al., 2009). Moreover, onthe basis of the complexity of the transport process, the largerange of substrate sizes, and the diversity of ABC proteins, it isprobable that additional structural substates/intermediatesexist besides the two conformations revealed by the availablecrystal structures (Kerr et al., 2010; Jin et al., 2012; Korkhovet al., 2012; Choudhury et al., 2014; Du et al., 2014; Srinivasanet al., 2014). Electron paramagnetic resonance and doubleelectron–electron resonance spectroscopy have been used toprobe the conformational dynamics of ABC transporters (ZouandMcHaourab, 2010; Schultz et al., 2011a,b; Rice et al., 2013;Joseph et al., 2014; Mishra et al., 2014; Sippach et al., 2014 ).One study determined that the histidine ABC transportersampled open, semi-open, and closed conformations (Sippachet al., 2014). The closed conformation of the NBDs was onlyachieved in the presence of the ligand. Another study useddouble electron–electron resonance to probe conformationalchanges of the transport for the Escherichia coli vitamin B12ABC importer BtuCD-F. Both substrate and ATP wererequired for the closure of the cytoplasmic gate (Josephet al., 2014). Furthermore, structural changes of the antigen-processing protein, detected by fluorescence resonance energytransfer (FRET), revealed that substrate is required for thecomplete NBD closure (Geng et al., 2013). Finally, association/dissociation of MsbA NBDs studied by luminescence resonanceenergy transfer revealed that the NBDs separate completelyafter each hydrolysis cycle (Cooper and Altenberg, 2013).To investigate the dynamics of the human MRP1 trans-

porter under physiologic conditions in the native membrane ofa living cell, we engineered a “two-color MRP1” construct withfluorescent protein tags fused to the C-terminal side of NBD1and NBD2 (Fig. 1). We predicted that changes in MRP1conformation would result in changes in the distance betweenthe green fluorescent protein (GFP) donor and the TagRFPacceptor, altering efficiency of intramolecular FRET. Specif-ically, a conformational change that brings the nucleotide-binding domains closer together was expected to increaseFRET from a basal level to a new higher level, reflecting thecloser proximity of the fluorescent tags. We have previouslyused this approach to monitor structural dynamics ofa calcium transporter within the native environment of thecell membrane (Hou et al., 2012; Pallikkuth et al., 2013). Thisstrategy may be of general utility for investigating theconformational changes of transporter proteins in the nativeenvironment of the live cell.

Materials and MethodsNucleotides, ATP analogs, doxorubicin (DOX), anti-MRP1 anti-

body, epigallocatechin gallate, mesalamine, calcipotriol, meropenem,benzamidine, phenylmethylsulfonyl fluoride, dithiothreitol, creatine

kinase, poly-D-lysine, saponin, and 2-mercaptoethanol were purchasedfrom Sigma-Aldrich (St. Louis, MO). [6,7-3H]E217bG (45 Ci mmol–1)was purchased from PerkinElmer (Waltham, MA). E217bG, creatinephosphate, and sodium orthovanadate were from Santa CruzBiotechnology (Dallas, TX). MK571 [3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid] was received from Cayman Chemical (Ann Arbor,MI) and bismaleimidohexane (BMH) was from Pierce Biotechnology(Rockford, IL). Restriction enzymes were purchased from NewEngland Biolabs (Ipswich, MA) and phosphate-buffered saline waspurchased from Thermo Scientific (Sunnyvale, CA).

Engineering Two-Color MRP1. Using pTagRFP-N vector asa backbone, we generated a two-color human MRP1 construct. First,C-terminal 2-kb fragment of MRP1 encoding amino acids 874–1531(stop codon removed) was generated using primers (forward, 59 GTACCG CGG CAG GAG CAG GAT GCA GAG GAG AAC 39; reverse,59 GTA ACC GGT CT CAC CAA GCC GGC GTC TTT GGC CAT G 39)and cloned in frame with TagRFP. Engineered restriction sites areunderlined. In the second step, 0.7-kb fragment encoding GFPproduced using primers (forward, 59 GTA GTC GAC ATG GTG AGCAAG GGC GAG GAG 39 ; reverse, 59 GTA CCG CGG CTT GTA CAGCTC GTC CAT GCC 39) was fused to the construct. Finally,N-terminal, 2.6-kb fragment of MRP1 encoding amino acids 1–873generated with primers (forward, 59 CTA GAG CTC ATG GCG CTCCGG GGC TTC TGC 39; reverse, 59 GTA GTC GAC CTC TGT GCTGGC ATA GGT ACG CAG GAA 39) was ligated in frame with GFPto produce the final construct. The integrity of the sequence wasconfirmed by sequencing. In the final two-color MRP1 recombinantprotein construct, the intrasequence GFP is flanked by two amino acidlinkers on the N-terminus (Val-Asp) and C-terminus (Pro-Arg). Thestop codon of MRP1 was removed and TagRFP was fused to theC-terminal end of MRP1 with a five–amino acid linker (Thr-Gly-Leu-Ala-Thr). The schematic diagram of two-colorMRP1 is shown inFig. 1A.Positions of GFP and TagRFP insertion were chosen to reflect themovements of NBDs and were guided by available crystal structuresand sequence alignment analysis. Closure of the NBDs was predicted toresult in a change from a low-FRET state to a high-FRET state. Theother major consideration in the design was to avoid regions critical forthe expression and function of MRP1.

MRP1-Expressing Stable Cell Line. Two-color MRP1 expres-sion vector was transfected into AAV-293 cells (Agilent, Santa Clara,CA) using jetPRIME Transfection Reagent (VWR, Radnor, PA),according to the manufacturer’s instructions. After 24 hours, stan-dard medium was replaced with medium containing G418 (400 mg/ml).Cells were maintained under G418 selection for 2 weeks. Then, the G418amount was increased to 800 mg/ml. GFP-expressing cells were sortedfrom the nonexpressing cells by flow cytometry and maintained underG418 selection of 200 mg/ml.

Cell Culture, Immunoblots, and Fluorescence Microscopy.MRP1 expression vectors were transiently transfected into AAV-293cells using jetPRIME transfection reagent according to the manu-facturer’s instructions. MRP1 proteins were detected by immunoblotanalysis using the mouse monoclonal anti-MRP1 antibody (dilution1:5000). Primary antibody binding was visualized by enhancedchemiluminescence detection using goat anti-mouse horseradishperoxidase–conjugated secondary antibody (dilution 1:10,000). Fluo-rescence microscopy of AAV-293 cells expressing two-color MRP1 wasperformed in glass-bottom chambered coverslips (Matek Corporation,Ashland, MA) coated with poly-D-lysine 24–36 hours post-transfectionwith jetPRIMEmammalian transfection kit. Fluorescent images werecollected using a Nikon inverted microscope (TE 2000-U; Tokyo,Japan) equipped with a 60� oil objective. Widefield lamp excitationwas used for FRET measurements, using computer-controlled filterwheels, as previously described (Hou et al., 2008; Kelly et al., 2008;Hou and Robia, 2010; Bidwell et al., 2011; Ha et al., 2011; Song et al.,2011). To determine the effect of substrate and/or ATP on MRP1structure, cells were permeabilized for 2 minutes in 50 mg/ml saponinin phosphate-buffered saline (PBS). Saponin permeabilization provides

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access for small molecules in the bath and provides control over theintracellular concentration of ATP and other impermeant species(Bidwell et al., 2011; Hou et al., 2012; Abrol et al., 2014). The saponin-containing buffer was removed and cells were rinsed once with PBS,followed by solution replacement with PBS containing substrate and/orATP replacing endogenous nucleotides that diffused out of the cells intothe large volume in the bath.

Confocal microscopy was performed using a Leica (Wetzlar,Germany) SP5 confocal microscope equipped with a 1.3 numericalaperture 63� water-immersion objective. Excitation was accom-plished with argon laser illumination at 488 nm for GFP and DOX,with emission bands of 496–533 nm for GFP and 565–650 for DOX.Total internal reflection fluorescence (TIRF) microscopy was per-formed with a Nikon invertedmicroscope (Ti-E) equipped with a 100�oil-immersion objective (numerical aperture 5 1.49) and a cooledcharge-coupled device camera (iXon 887; Andor Technology, SouthWindsor, CT). Through-objective TIRF excitation was achieved witha 488-nm argon laser (for GFP). The laser incident angle was adjustedto create an evanescent field that illuminated the plasma membranein contact with the surface substrate. Fluorescence recovery afterphotobleaching (FRAP) was performed as previously described (Robiaet al., 2007; Bossuyt et al., 2011). For spatially resolved photo-bleaching, the argon laser 488-nm line was selected by a laser linefilter and directed to the sample with a 10/90 beam splitter. Thebleach beam was focused to a 1.3-mm target region on the specimenusing a Keplerian telescope composed of two planoconvex lenses.Laser photobleaching exposure time was controlled by a Uniblitzshutter and was 100 milliseconds. Filter transitions and shutterevents were automated to provided reproducible timing. Image datawere quantified with MetaMorph analysis software (MolecularDevices, Sunnyvale, CA).

Transport of [3H]E217bG by MRP1-Enriched MembraneVesicles. Wild-type and two-color MRP1 expression vectors weretransfected into AAV-293 cells (at 50–70% confluency). After 48 hours,the cells were harvested, and membrane vesicles were prepared asdescribed previously (Iram and Cole, 2014). The total protein contentof the vesicles was quantified using a Bio-Rad Bradford assay(Hercules, CA) with bovine serum albumin as a standard. ATP-dependent transport of [3H]E217bG into the inside-out membranevesicles was measured by a rapid filtration technique (Loe et al.,1996). In brief, 10 mg of membrane vesicle protein was incubated with400 nM/120 nCi [3H]E217bG for 1 minute at 37°C in a 100-ml reactionmixture containing 10 mM MgCl2 and 4 mM AMP or 4 mM ATP intransport buffer (250 mM sucrose and 50 mM Tris-HCl, pH 7.4).Reactions were stopped by dilution in ice-cold transport buffer,followed by filtration through glass fiber filters. Filters were washedwith the transport buffer and radioactivity associated with thevesicles was determined. ATP-dependent uptake was calculated bysubtracting the uptake in the presence of AMP from the uptakemeasured in the presence of ATP. Transport assays were carried outin triplicate, and results were expressed as means 6 S.D. andcorrected for any differences in expression of MRP1.

FRET Measurement. FRET was quantified from cells attachedto polylysine-coated glass coverslip chamber dishes as previouslydescribed (Hou and Robia, 2010; Bidwell et al., 2011; Hou et al., 2012),using the E-FRET acceptor sensitization method (Zal and Gascoigne,2004). Fluorescent images were analyzed using MetaMorph software,with fluorescence intensities quantified on a cell-by-cell basis. FRETefficiency (E) was calculated according to the relationship

E5IDA 2aðIAAÞ2dðIDDÞ

IDA 2aðIAAÞ1 ðG2dÞðIDDÞ (1)

where IDD is the intensity of fluorescence emission detected in thedonor channel (520/28-nm) with excitation of 480/17 nm; IAA isacceptor channel (617/73-nm) emission with excitation of 556/20 nm;IDA is the FRET channel, with 617/73-nm emission and excitation of480/17-nm; a and d are cross-talk coefficients determined from

acceptor-only or donor-only samples, respectively.We obtained valuesof d5 0.165 (for GFP) and a5 0.047 (for TagRFP).G is the ratio of thesensitized emission to the corresponding amount of donor recovery,which was 1.82 for this setup. Probe separation distance (R) was cal-culated from the relationship described byFörster,R5 (R0)[(1/E) – 1)

1/6,where E is the measured FRET value and R0 is the Förster radius,which is 58.3 Å for the GFP-TagRFP pair. This distance calculationincludes the widely used assumption of random relative orientation ofthe donor and acceptor fluorescence dipoles (k2 5 2/3). Failure of thisassumption would alter the quantitative value of the distancecalculated but is not expected to alter the biologic conclusions of thisstudy, which are drawn from relative changes in FRET. The reportedFRET efficiency value represents the mean of FRET efficienciesobtained from 75 to 125 cells. For comparison of FRET with proteinexpression level, we quantified cell-by-cell TagRFP fluorescence (inarbitrary units) as an index of MRP1 expression.

High-Throughput Screening. Cells were transfected using293fectin (Invitrogen/Life Technologies, Carlsbad, CA) and G418(Sigma-Aldrich) selection was used to select stable cell lines overa period of 2–3 weeks. Fifteen cell lines expressing two-color MRP1were further selected by fluorescence microscopy and flow cytometry.The optimal cell line was .98% positive for GFP fluorescencemeasured by flow cytometry. Screening of the NIH Clinical Collection(NCC; a library of 446 drug-like compounds) was performed aspreviously described (Gruber et al., 2014). The NCC is a library ofunique compounds that is ideal for small-scale trial screens. Thecompounds are potential drug candidates that have been used inhuman clinical trials and are well defined with respect to safetyprofile, molecular weight, and chemical properties. All compoundswere dissolved in dimethyl sulfoxide (DMSO), and DMSOwas presentin all control wells. Cells stably expressing two-color MRP1 wereremoved from a 225-cm2 flask by incubating with TrypLE (Invitrogen/Life Technologies) for 5 minutes. Cells were collected and pelleted bycentrifugation at 500g for 5 minutes, followed by resuspension in 10 mlof PBS. Cell density was determined using a Countess cell counter(Invitrogen/Life Technologies), and cells were diluted to 1� 106 cells/ml.Forty-nine milliliters of the cell solutions was plated by hand intoa 384-well plate containing NCC compounds using a 12-channelmultipipet. Assay plates were spun for 1 minute at 200g and allowedto incubate at room temperature for 20 minutes before being readon a NovaFluor fluorescence lifetime plate reader (FluorescenceInnovations, Inc., Minneapolis, MN). GFP fluorescence was excitedwith a 473-nm microchip laser from Concepts Research Corpora-tion (Belgium, WI), and emission was filtered with 490-nm longpass and 520/35-nm band pass filters from Semrock (Rochester,NY). Time-resolved fluorescence waveforms for each well werefitted to singly-exponential decays (single-lifetime model) usingleast squares minimization global analysis software (FluorescenceInnovations, Inc.) Compounds that did not possess intrinsic fluores-cence and that changed the fluorescence lifetime by more than threestandard deviations were identified as hits. Dose-response assays wereobtained for four of the eight NCC library compounds identified ashits. Each compound was obtained from Sigma-Aldrich, prepared at10 mM in DMSO, then further diluted in DMSO to the appropriateworking concentration. Each compound was plated in three wells of a384-well plate, cells were added, and the two-color MRP1 fluorescencelifetime was quantified as described above.Wemeasured Z9 values foreach of the four assay plates included in this study, as described in thehigh-throughput screening Assay Guidance Manual (http://www.ncbi.nlm.nih.gov/books/NBK53196/). Our data for GFP alone (donor only)comes from a screen of GFP-SERCA2a of the same NCC library on thesame day as the first NCC screen of 2C-MRP1. The Z9 values for eachplate were .0.5 (average 5 0.64), exceeding the NIH-recommendedstandard of 0.4. We also measured the coefficient of variance (CV) forthe control wells on each plate to assess instrument precision andensure that the cells had been plated uniformly. Inclusion criteria wasa CV , 1.5% for each plate, and average CV for the four NCC assayplates was 1.1%.

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To determine whether MRP1 function was altered by hit com-pounds identified in the high-throughput screen, we measuredDOX extrusion in MRP1-expressing cells. Five hours after platingon glass-bottom chambered coverslips, transiently transfectedcells were preincubated for 15 minutes with 10 mM candidatecompound, then 2 mM DOX was added for 1 hour. Cells wereimaged by confocal microscopy and the red fluorescence in thenucleus was quantified as an index of the amount of DOXaccumulated in the nucleus. Cells expressing MRP1 were com-pared with untransfected cells in the same microscopic field.Approximately 100 transfected and 100 untransfected cells werequantified for each compound. Comparisons between transfectedcells and untransfected cells and between different compoundswere evaluated by one way analysis of variance, with �Sídák’smultiple comparisons test.

Results and DiscussionExpression, Localization, and FRET of Two-Color MRP1

To investigate the structural dynamics of MRP1 in itsnative membrane environment, we engineered a two-colorMRP1 construct by fusing GFP and TagRFP at theC-terminus of NBD1 and NBD2, respectively (Fig. 1A), andquantified FRET changes as an index of NBD conformationalchanges. MRP1 expression was verified by immunoblotanalysis using cell lysates of AAV-293 cells transfected withcDNA expression vectors encoding wild-type or two-colorMRP1, loading 10 mg of protein per lane. The recombinantMRP1 proteinmigratedmore slowly than the untaggedMRP1and produced a band at the expected molecular mass of∼250 kDa (Fig. 1C). Another band of lower mobility was alsodetected, possibly the result of protein aggregation duringSDS-PAGE sample preparation. However, no aggregationwas apparent in intact cells. Two-color MRP1 showed theexpected plasma membrane localization by widefield fluo-rescence microscopy (Fig. 1D) and TIRF microscopy (Fig. 1E),with no bright puncta or other nonuniformities. Anotherprobable possibility is that the multiple bands representdifferential denaturation of the unboiled MRP1 protein. Inparticular, we have frequently observed that fluorescentprotein fusion tags are resistant to SDS and show mixedpopulations of denatured and undenatured species withdifferent PAGE mobilities. FRAP showed that two-colorMRP1 protein was readily diffusible in the plasmamembrane with an apparent diffusion coefficient of4.4 � 1023 mm2/s (Fig. 1F). This value is comparable todiffusion coefficients reported for a related ABC transporter,cystic fibrosis transmembrane conductance regulator (CFTR),as measured by FRAP (1.7 � 1023 mm2/s), image correlationspectroscopy (6.3 � 1023 mm2/s) (Bates et al., 2006), andsingle-particle tracking (4.0 �1023 mm2/s) (Jin et al., 2007).In permeabilized AAV-293 cells transiently transfectedwith two-color MRP1, we observed ∼17% FRET efficiencyirrespective of protein expression levels (Fig. 1G). The lack ofconcentration dependence is consistent with intramolecularFRET (Hou et al., 2012) with little apparent contribution ofnonspecific energy transfer (Ha et al., 2011) or otherintermolecular FRET. Although we cannot formally rule outthe possibility of formation of MRP1 oligomers, the datasuggest that the oligomeric status of MRP1 remained thesame over the range of protein concentration used in theseexperiments.

MRP1 Transport Activity in Membrane Vesicles and LiveCells

To determine if the engineered two-color MRP1 wasfunctional, the vesicular transport activity was measuredusing a prototypical MRP1 substrate, E217bG. Inside-outmembrane vesicles prepared from AAV-293 cells transientlytransfected with wild-type or two-color MRP1 were used todetermine the uptake of this organic anion. Two-color MRP1membrane vesicles exhibited similar levels of [3H]E217bGuptake as those of wild-type MRP1 membrane vesicles (Fig.2A). Results were comparable to other reports (Iram and Cole,2012, 2014).Two-color MRP1 transport activity was also evaluated in

live cells by measuring extrusion of the fluorescent anticancerdrug DOX, a well known substrate of MRP1. Confocalmicroscopy of AAV-293 cells showed DOX accumulating in thenucleus of untransfected cells (Fig. 2B, red), but DOXfluorescence was not observed in cells expressing two-colorMRP1 (Fig. 2B, green). These results indicate that the two-color MRP1 trafficked properly to the plasma membrane andwas functionally active as an efflux pump. MRP1-dependentDOX efflux was blocked by the known MRP1 inhibitorMK-571 and by BMH, a cell-permeable cross-linker (Rajagopaland Simon, 2003) (Fig. 2B). The present results are not consis-tent with a previous study that showed intracellular localiza-tion of MRP1. That report suggested that drug transportersMRP1, P-gp, and BCRP do not reduce the overall accumulationof doxorubicin within the cell; rather, they confer multidrugresistance by sequestering the drug in intracellular vesicles,sparing the nucleus (Rajagopal and Simon, 2003). Here we seeclear and uniform expression of two-color MRP1 mostly at theplasma membrane (Fig. 2B, green). These data are consistentwith a model in which ABC drug transporters such as MRP1,P-gp, and BCRP function as plasma membrane efflux pumps,conferring multidrug resistance by reducing the intracellularaccumulation of the drug (Aller et al., 2009; Iram and Cole,2014).

MRP1 Conformational Changes

To measure ligand-dependent conformational changes ofMRP1 protein, we performed FRET microscopy of saponin-permeabilized (Ruggiero et al., 1985; Wassler et al., 1987;Jamur and Oliver, 2010) cells, which provided control over theconcentration of nucleotide and transportable substrates bywashing out the endogenous contents, including the nucleo-tides. First, the effect of ATP binding on NBDs conformationwas investigated by measuring FRET changes at differentATP concentrations. As seen in Fig. 3A, FRET increased withATP in a concentration-dependent manner to a maximumFRET of 25% at 1mMATP. The final value was similar to thatmeasured in intact (unpermeabilized) cells. The data suggestthat ATP binding induces closure of the MRP1 cytoplasmicdomains, bringing opposing NBDs into close proximity. Ahyperbolic fit to the data gave an apparent ATP bindingaffinity of 107 mM. The ATP concentration dependence of theconformational change did not appear to be positivelycooperative, though other studies have provided evidence forcooperativity of ATPase activity with MRP1 and other ABCtransporters (Senior and Bhagat, 1998; Ramaen et al., 2006;Zaitseva et al., 2006). A Hill fit of the data in Fig. 3A yieldeda Hill coefficient of 0.72 (error of fit 0.45). The average FRET

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observed for two-color MRP1 in apo condition was 16.7 61.0%, and FRET did not change significantly with the additionof the substrate (E217bG) (P 5 0.20), suggesting that therewas not a detectable conformational change in the NBDs uponsubstrate binding (Fig. 3B). These results are consistent withthe crystal structure study, which showed that both apo andthe inhibitor-bound P-gp structures were almost identical(Aller et al., 2009). In contrast, the presence of 1 mM ATPalone led to a significant increase in FRET to 25.36 0.6% (P50.0002 versus apo), consistent with a conformational changethat decreased the distance between the donor and acceptorfluorescent tags. Even though substrate alone did not induceNBD closure, E217bG added in the presence of ATP increasedFRET to 28.6 6 1.3% (P 5 0.019 versus ATP alone) (Fig. 3B).We observed a similar response for another known MRP1substrate, cysteinyl leukotriene (P5 0.022 versus ATP alone).The order of addition of substrate and ATP did not affect thefinal FRET efficiency. Our data suggest that ATP bindinginduces the major conformational change and brings thetwo NBDs closer; however, substrate binding is required toachieve more compact, high-FRET conformation. Notably,FRET was not increased by AMP and only modestly elevatedby ADP. This is in contrast to a recent study of P-gp (Verhalenet al., 2012) that reported an incremental increase in intra-molecular FRET efficiency with the addition of substratealone.

Interpretation and Limitations of FRET Measure-ments. FRET distances were calculated from the Försterrelationship, assuming random relative dipolar orientation(k2 5 2/3). Deviations from this assumption would increase ordecrease the apparent probe separation distance. Moreover,the expressed fluorescent tags are large (∼29 KDa), whichadds uncertainty about the relative position of the tag and theunderlying domain. However, our recent computational studyprovides support for the assumption that fluorescent proteinscommunicate closely with the domain to which they are at-tached, and thus FRET changes may serve as a useful index ofprotein conformational changes (Smolin and Robia, 2015).Here, nonspecific FRET was not subtracted from the mea-sured FRET values (Kelly et al., 2008; Ha et al., 2011) becausethere was no evidence of a concentration-dependent increasein FRET (Fig. 1G). Overall, our results indicate that the GFPand TagRFP are separated by 76.2 6 0.9 Å in the apocondition, and upon binding to ATP and ATP/substrate thisdistance was decreased to 69.8 6 0.4 and 67.9 6 0.7 Å,respectively. The apparent magnitude of the NBD movementobserved here was less than would be predicted fromcomparison of the mouse P-gp (apo) and nucleotide-boundSav1866 crystal structures (∼20 Å difference) (Dawson andLocher, 2006; Aller et al., 2009). However, it is important tonote that ensemble measurements of average FRET efficiencymay overlook multiple structural subpopulations (Verhalen

Fig. 2. Functional characterization of two-color MRP1. (A)ATP-dependent uptake of [3H]E217bG by the membranevesicles. Membrane vesicles were incubated with substrateand transport reaction was allowed for 1 minute at 37°C.Values are mean 6 S.D. for triplicate determinations ina single experiment; both wild-type (WT-MRP1) and two-color MRP1 (2C-MRP1) values were significantly differentfrom control (P , 0.05). Similar results were obtained ina second experiment with vesicles derived from indepen-dent transfection. (B) AAV-293 cells (control) and two-colorMRP1-expressing cells were exposed to 10 mM DOX for15 minutes. DOX-containing media was replaced with PBS,and cells were imaged using a confocal microscope equippedwith 63� objective. DOX and GFP were excited at 488-nmwavelength using an argon laser, with emission bands of565–650 nm for DOX and 496–533 nm for GFP. DOX wasdetected by red fluorescence and was mostly localized in thenucleus; two-color MRP1 (green) showed plasma membranelocalization. To inhibit MRP1 activity, cells were incubatedwith MK571 (50 mM) or BMH (1 mM) for 15 minutes beforeDOX treatment. All error bars represent mean 6 S.E.

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Fig. 3. Dynamic FRET changes of two-color MRP1 in permeabilized AAV-293 cells. FRET measurements were made as described in the experimentalprocedures. Cells plated on glass-bottom–chambered coverslips were permeabilized for 2 minutes at room temperature in 50 mg/ml saponin in PBS,which washed out the endogenous cell contents including the nucleotides. PBS buffer supplemented with 5 mM MgCl2 was used in all FRETexperiments. All incubations (10–15 minutes) and FRET measurements were made at room temperature. (A) FRET increased with ATP. (B) MRP1FRET response to nucleotide and transportable substrates E217bG or cysteinyl leukotriene (LTC4). (C) MRP1 interaction with ATP analogs applied at1 mM concentration. (D) Competition of 1 mM nucleotide analogs with 500 mMATP. Analogs did not prevent ATP from increasing MRP1 FRET. (E) ATPanalogs increased MRP1 FRET when applied at high concentration. (F) The effect of inhibitors of MRP1, including 1 mM vanadate, 50 mM MK571, and1 mM BMH. With the exception of the apo condition, 1 mM ATP + 50 mME217bG were added to each. ATP, ATP+ E217bG, ATP+LTC4, and ADP valueswere statistically significant versus apo (P , 0.05). All error bars represent mean 6 S.E. AMP-PCP, adenylylmethylenediphosphonate.

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et al., 2012; Pallikkuth et al., 2013). Moreover, substratebinding may not induce an all-or-nothing switch to a newconformation but rather shift the balance of an equilibrium ofmultiple structures. Thus, the apparent probe separationdistance change of ∼8 Å measured here may underestimatethe true magnitude of the MRP1 conformational change.

MRP1 Interaction with ATP Analogs

Structural studies have obtained high-quality crystals bytrapping ABC transporters in a prehydrolysis state byincubation with nonhydrolyzable ATP analogs (Dawson andLocher, 2007). Here we quantified the structural change ofMRP1 in response to ATPgS, adenylylimidodiphosphate(AMP-PNP), and adenylylmethylenediphosphonate. All of theanalogs produced a much smaller FRET increase comparedwith ATP (Fig. 3C). This was unexpected since these analogsare generally assumed to have a binding affinity that is ashigh as ATP. One possibility is that the analogs bind tightlybut do not induce a significant closure of the NBDs. If so, theanalogs would be expected to compete with ATP for thenucleotide-binding site. However, incubation of MRP1 with1 mM analog did not prevent 500 mM ATP from increasingFRET to 28–29% versus apo-MRP1 (Fig. 3D). The datasuggest that ATP analogs do not prevent closure of MRP1,since they do not compete with ATP and reduce FRET. Rather,ATP analogs may bind with poor affinity to NBDs of MRP1.This interpretation is supported by the observation that higherlevels of the ATP analogs (5mM) did increase FRET (Fig. 3E) tovarying degrees, all somewhat less potently than ATP. ATPanalogs AMP-PNP and adenylylmethylenediphosphonate inparticular showed a reduced maximal FRET. It is unknownwhether these nucleotide analogs stabilize the same MRP1conformation as ATP. High-resolution crystal structures ofa related bacterial transporter, Sav1866, showed that AMP-PNP produced an outward-facing conformation that was notgrossly different from the ADP-bound structure (Dawson andLocher, 2006, 2007).

Inhibition of MRP1 Activity

ATPases can be arrested in a posthydrolysis state byincubation with orthovanadate under conditions permissivefor ATP hydrolysis (Iram and Cole, 2011), trapping ADP atthe catalytic site (Koike et al., 2004). When orthovanadate-induced ADP trapping was performed in AAV-293 cellspermeabilized with saponin, the FRET value obtained was29.2 6 0.7%, similar to the FRET value for the activetransport condition in the presence of ATP and substrate,28.7 6 0.9% (Fig. 3F). The data suggest that theorthovanadate-induced transition state also increases thepopulation of molecules in the most compact conformation.The closely interacting NBDs of the ADP-trapped transporterimply that resetting of the transporter to an open (low FRET)conformation is accomplished by release of the substrate and/or ADP rather than ATP hydrolysis. A recent study (Verhalenet al., 2012) focused on P-gp NBD structural dynamicsreported a significant increase in FRET upon addition ofinorganic vanadate, but we observed no such effect ofvanadate with MRP1. As with orthovanadate, MRP1 in-hibition by MK571 caused no significant change in theaverage FRET efficiency (Fig. 3F) compared with the activetransport condition. The data suggest that MK571 stops

enzymatic cycling by stabilizing a high-FRET conformationof MRP1. This is consistent with the proposed mechanismof competitive inhibition of substrate binding by MK571(Gekeler et al., 1995) However, crosslinking the transporterwith BMH reduced intramolecular FRET from 28.7 to 22.4%(Fig. 3F). This is consistent with BMH crosslinking MRP1 inan intermediate conformation, partway between apo (open)and ATP-bound or substrate-bound (closed) structures.

Potential Application for Drug Discovery

Previously, we used a different two-color pump, the P-typeATPase SERCA, to detect activator/inhibitor candidates ina high-throughput screen of a library of drug-like compounds(Gruber et al., 2014). The present data suggest that thisapproach can be successfully generalized to ABC proteinsand to other families of transporters. To evaluate whethertwo-color MRP1 could be useful for identification of novelsubstrates or inhibitors, we performed a FRET-based screenof the NCC library of compounds that are known to bebiologically active. We applied test compounds to live cells ina 384-well assay format as previously described (Gruber et al.,2014) and selected hits that altered the fluorescence lifetimeof the GFP donor on MRP1 in repeated screens of the library.Two independent screens identified 20 and 34 hits, re-spectively, with 13 hits in common between the two screens.Of the 13, eight were in the normal range of intensity.Notably, several of these compounds are very closely relatedstructurally (Fig. 4A); calcipotriol is an analog of calcitriol andepigallocatechin gallate is very similar to hyperoside. Figure 4Bshows that the fluorescence lifetime provided a more robustselection criterion than fluorescence intensity, with betterwell-to-well reproducibility and improved signal-to-noiseratio. We selected four of the eight hits for additionalcharacterization. Figure 4C shows the dose response forepigallocatechin gallate (antioxidant), mesalamine (anti-inflammatory), calcipotriol (vitamin D analog), and merope-nem (antibiotic). Whereas meropenem and mesalamineproduce only weak effects on donor lifetime, both epigallo-catechin gallate and calcipotriol elicited large lifetimechanges with sigmoidal dose dependence. Epigallocatechingallate, in particular, showed a high apparent affinity (EC50 51.7 mM) with evidence of cooperative binding (Hill coefficient52.06). To test whether candidate compounds altered thetransport activity of MRP1 we quantified DOX accumulationin the nuclei of human embryonic kidney cells as in Fig. 2B.Representative images of cells for each treatment condition areprovided in Supplemental Fig. 1. Figure 4D shows that DOXaccumulation in the nuclei of cells was greatly reduced byexpression of MRP1 (“1MRP1”, *P , 0.0001 versus untrans-fected), but pretreatment with known MRP1 inhibitor MK571(1 mM) decreased this extrusion activity (Fig. 4D; #P , 0.0001versus “1MRP1”), increasing DOX fluorescence in the nucleus(Supplemental Fig. 1). We could not evaluate the effect ofmesalamine because it interfered with the DOX treatmenteven in untransfected cells. Supplemental Fig. 1 shows thatcells pretreated with mesalamine showed DOX fluorescencelocalized to intracellular compartments but not in the nucleus,irrespective ofMRP1 expression. The reason for this interferencewith DOX uptake was unclear. No apparent decrease intransport activity was observed for epigallocatechin gallateor meropenem, but calcipotriol decreased MRP1 activity,

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increasing DOX accumulation (Fig. 4D, #P 5 0.0085 versus“1MRP1”). The data suggest that calcipotriol (vitamin Danalog) may interfere with the ability of MRP1 to transportDOX. This result is noteworthy as vitamin D has beenobserved to potentiate the antitumor activity of DOX andother chemotherapeutic agents (Ma et al., 2010).The data demonstrate the potential utility of MRP1 intra-

molecular FRET for discovery of small-molecule modulators ofMRP1 structure/function. This assay offers several advan-tages over alternatives. Comparedwith transport assays (Fig. 2,A and B), a FRET-based screen is simple, it does not involveaddition of other compounds (like DOX), and it does not requirea wash step or detailed calibration. It is also potentiallysensitive to both allosteric regulators and transportable sub-strates, whereas an assay on the basis of quantifying transportmay not detect substrates that bind weakly or fail to competewith the test substrate. Future studies will screen largerlibraries of compounds to identify additional molecules thatalter MRP1 intramolecular FRET. Potential hit compoundscould include transportable substrates or novel inhibitors ofMRP1 activity.

ConclusionsWe have engineered a two-color MRP1 protein construct

that exhibits large changes in FRET in response to binding ofnucleotide, transportable substrates, and inhibitory drugs. Inaddition to performing well in a pilot high-throughput screenof a drug library, the construct has provided insight into theconformational changes during the transport cycle of MRP1.The data are broadly compatible with the ATP switch model(Abele and Tampe, 2004; Higgins and Linton, 2004), in whichtransport is driven by NBD association/dissociation in re-sponse to ATP binding and hydrolysis. However, given theuncertainties of the present ensemble FRET measurements,we cannot rule out alternative models, such as constant-contact models in which the NBDs do not fully dissociate butopen enough to allow ADP/ATP exchange. Moreover, it isprobable that additional conformational states exist besidesthe two states detected by crystallography. Nevertheless, thedata suggest that intramolecular FRET is a useful index ofthe conformational changes of transport and underscore theutility of fluorescent protein tags as reporters of pumpdynamics. They are generally benign for function (Bidwell

Fig. 4. High-throughput screening with two-color MRP1.(A) Hits from the National Clinical Collection. (B) Weobserved significantly less variability and improved signalto noise when screening with two-colorMRP1 on the basis offluorescence lifetime compared with fluorescence intensity.(C) Dose response for select hit compounds. Epigallocate-chin gallate showed the highest apparent affinity (EC50 =1.7 mM). (D) DOX uptake into the nucleus of humanembryonic kidney cells after pretreatment with candidatecompounds. Values are mean nuclear fluorescence 6 S.E.,normalized to untransfected cells in the same microscopicfield. The red horizontal line indicates a high level of DOXaccumulation (as in untransfected cells), the green lineindicates decreased accumulation of DOX suggestingdrug efflux by MRP1. *P , 0.0001 versus untransfected;*P , 0.0001 versus “1MRP1”. All error bars representmean 6 S.E.

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et al., 2011; Hou et al., 2012) (Fig. 2) and can be fused toarbitrary positions with fixed stoichiometry for maximumsensitivity to the conformational change of interest. To scalethis assay for high-throughput screening, two-color trans-porters may be expressed in stable cell lines (Gruber et al.,2012), allowing selective retrieval of candidate compoundsthat are nontoxic, cell permeant, and potent for eliciting thedesired conformational change. For two-color MRP1, inparticular, we envision a discovery program that may identifyexisting drugs not previously known to be transported by thisbroad spectrum drug-resistance protein. Such informationmay provide insight into previously unexplained drug re-sistance or drug interaction mechanisms. MRP1 screeningmay also yield novel inhibitors useful for treating chemother-apy resistance in anticancer regimens.

Acknowledgments

The authors thank Daniel J. Blackwell, Zhanjia Hou, Ryan D.Himes, and Nikolai Smolin for technical assistance. The authors alsothank Prof. Toni Pak for assistance with radioisotope transportassays.

Authorship Contributions

Participated in research design: Iram, Robia, Thomas.Conducted experiments: Iram, Raguimova, Gruber.Contributed new reagents or analytic tools: Iram, Robia, Thomas.Performed data analysis: Iram, Robia, Gruber, Thomas, Raguimova.Wrote or contributed to the writing of the manuscript: Iram, Robia,

Gruber, Thomas.

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