STRUCTURAL BIOLOGY

Molecular structure of humanP-glycoprotein in the ATP-bound,outward-facing conformationYoungjin Kim and Jue Chen*

The multidrug transporter permeability (P)–glycoprotein is an adenosine triphosphate(ATP)–binding cassette exporter responsible for clinical resistance to chemotherapy.P-glycoprotein extrudes toxic molecules and drugs from cells through ATP-poweredconformational changes. Despite decades of effort, only the structures of the inward-facing conformation of P-glycoprotein are available. Here we present the structure ofhuman P-glycoprotein in the outward-facing conformation, determined by cryo–electronmicroscopy at 3.4-angstrom resolution. The two nucleotide-binding domains form a closeddimer occluding two ATP molecules. The drug-binding cavity observed in the inward-facingstructures is reorientated toward the extracellular space and compressed to precludesubstrate binding. This observation indicates that ATP binding, not hydrolysis, promotessubstrate release. The structure evokes a model in which the dynamic nature ofP-glycoprotein enables translocation of a large variety of substrates.

Cancer cells develop resistance to chemicallydiverse compounds, a phenomenon knownas multidrug resistance (MDR). To im-prove the effectiveness of chemotherapy,many laboratories have searched for mech-

anisms that account for MDR. In 1973, Keld Danø

demonstrated that the reduced drug accumu-lation in tumor cells was energy dependent (1).In 1976, by labeling cell-surface carbohydrates,Juliano and Ling identified a glycoprotein en-riched in colchicine-resistant cells but not inwild-type cells (2). The protein was named thepermeability (P)–glycoprotein (Pgp) because itwas thought to confer drug resistance by makingthe cellular membrane less permeable (3). Tenyears later, the genes responsible for MDR in

human, mouse, and hamster (namedMDR genes)were cloned (4–6), and it was shown that the pro-tein product of themdr1 gene was indeed Pgp (7 ).Pgp is an adenosine triphosphate (ATP)–binding

cassette (ABC) transporter, which uses the ener-gy from ATP hydrolysis to pump substrates acrossthe membrane. It contains two transmembranedomains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs) (Fig. 1A). Pgp is ex-pressed in many membrane “barriers” of the body,including the blood-brain barrier, gastrointestinaltract, kidney, liver, ovary, and placenta (2, 8–11).Thus, the physiological function of Pgp is likelyto protect sensitive tissues and the fetus fromendogenous and exogenous toxicity (12). Morethan 300 compounds have been identified aspotential substrates of Pgp (13, 14). Drug resist-ance mediated by Pgp depends on ATP hydrol-ysis (15–17 ), and the adenosine triphosphatase(ATPase) activity of Pgp is stimulated by the trans-ported drugs (18–20). Vanadate (Vi) trapping andphotocleavage experiments showed that Pgp con-tains two active ATPase sites, but only one ATPis hydrolyzed at a time (21).The molecular structures of Pgp in a trans-

port cycle have been investigated by using avariety of methods, including antibody bind-ing (22), tryptophan fluorescence (23), lumines-cence (24), double electron-electron resonance(DEER) (25 ), electron microscopy (EM) (26, 27 ),and x-ray crystallography (28–31). Although thecrystal structures of Pgp have been determinedfor the mouse (28, 29), Caenorhabditis elegans(30), and Cyanidioschyzon merolae (31) ortho-logs, all of these structures exhibit a similar con-formation, in which the two NBDs are separated

RESEARCH

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Fig. 1. Characterization of recombinant human Pgp. (A) Topologydiagram of Pgp. Residues not resolved in the structure are shown as dashedlines. OUT, outer cell membrane; IN, inner cell membrane. (B) Vinblastinestimulates the ATPase activity of the wild-type (WT) protein but not theE556Q/E1201Q mutant (EQ). Data points represent the means ± standarddeviation of three to nine measurements at 29°C. By nonlinear regression ofthe Michaelis-Menten equation, WT Pgp in n-dodecyl-b-D-maltopyranoside(DDM) and cholesteryl hemisuccinate (CHS) solution has a Km of 30 ± 3 mM

for vinblastine and a maximal ATPase activity of 195 ± 8 nmol/mg/min.(C) The ATPase activity as a function of ATP concentration. In the presence of100 mM vinblastine, the WTprotein has a Km of 0.179 ± 0.05 mM for ATP anda maximal ATPase activity of 260 ± 16 nmol/mg/min. (D) The overallstructure of the E556Q/E1201Q Pgp in complex with ATP. The N-terminal half(TMD1 and NBD1) is colored in orange and the C-terminal half (TMD2 andNBD2) in blue. ATP is shown in ball-and-stick format (gray, carbon; red, oxygen;blue, nitrogen; orange, phosphorus), and Mg2+ is shown as a sphere (magenta).

Howard Hughes Medical Institute, The Rockefeller University,1230 York Avenue, New York, NY 10065, USA.*Corresponding author. Email: juechen@rockefeller.edu

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from each other and the translocation path-way is accessible from inside the cell (i.e., theinward-facing conformation). To reveal confor-mational changes that enable substrate translo-cation, we pursued the structure of human Pgpin an ATP-bound, outward-facing conformation.Pgp contains two active ATPase sites, each

comprising the Walker A and Walker B motifs ofone NBD and the Leu-Ser-Gly-Gly-Gln (LSGGQ)motif of the other NBD. At each ATPase site, ahighly conserved glutamate residue acts as thecatalytic base for ATP hydrolysis. Mutating eithercatalytic glutamate severely reduces ATPase ac-tivity; concurrent mutations at both positionstrap Pgp in an ATP-occluded form, likely with aclosed-NBD dimer (32–34). On the basis of thesedata, we generated a mutant of Pgp in whichboth catalytic glutamates (abbreviated E, at res-idue positions 556 and 1201) were replaced byglutamines (E556Q/E1201Q). Compared to thewild-type (WT) protein, whose ATPase activityincreases as a function of ATP concentrationand is stimulated by the substrate vinblastine, theE556Q/E1201Q mutant remains inactive over alarge range of ATP and vinblastine concentra-tions (Fig. 1, B and C).For cryo-EM studies, the mutant protein was

incubated with 150 mM vinblastine and 10 mMMg2+-ATP to promote ATP occlusion (32–34).A cryo-EM data set consisting of ~1 million par-ticles was collected and analyzed for structural

homogeneity. Three-dimensional (3D) classifica-tion using Relion (35) showed that most of theparticles exhibited an NBD-dimerized conforma-tion (fig. S1). Refinement of the best class pro-duced a map at 3.4-Å resolution (figs. S1 and S2),enabling us to build a nearly complete modelof Pgp except for the two termini and residues81 to 104 and 631 to 694, which form an extra-cellular loop and intracellular linker, respectively(fig. S3).The overall structure of human Pgp is sub-

stantially different from the previously deter-mined inward-facing conformations (Fig. 1D).Instead of enclosing an internal cavity, the trans-membrane (TM) helices pack closely in the mem-brane inner leaflet. The two cytosolic NBDs makeextensive contacts with each other, forming thetypical “head-to-tail” dimer characteristic of ABCtransporters (Fig. 2A). Two ATP molecules arebound at the dimer interface, interacting withthe Walker A motif of one NBD and the ABCsignature motif of the other NBD (Figs. 1D and2). This structure, largely consistent with whatis expected of an outward-facing conformation,also reveals several unanticipated features that ad-vance our understanding of the transport cycle.Biochemical studies have shown that mouse

Pgp containing alanine substitutions of bothcatalytic glutamates occludes maximally a singleATP (32, 36). Nucleotide trapping by Vi or be-ryllium fluoride also occurs at only one of the

two catalytic sites (37, 38). This suggests struc-tural asymmetry of the two ATPase sites in theoutward-facing conformation. However, the struc-tures of the two NBDs are nearly identical (super-position of all 246 a carbons in the NBDs yieldsa root mean square deviation of 0.5 Å) (fig. S4A).Furthermore, the cryo-EM densities for bothATPs are equally strong, and the ATP moleculesmake very similar contacts with nearby residues(Fig. 2B and fig. S4B).The discrepancy between the structural and

biochemical data can be explained by the dif-ferent experimental conditions. The cryo-EM gridswere prepared with saturating concentrationsof ATP (10 mM); in the biochemical study, thestoichiometry values were determined after affin-ity chromatography in an ATP-free buffer (32, 36).Cryo-EM 3D classification analysis shows that, inthe presence of 10 mM ATP, about 5% of particlesreside in the inward-facing (non–ATP-occluded)conformation (fig. S1). Two other independentEM studies at low resolution have also shownthat only a fraction of Pgp molecules could betrapped in the outward-facing conformationby nonhydrolyzable ATP analogs or ATP with Vi(26, 27 ). Thus, it is possible that, in biochemicalstudies, a large fraction of the molecules convertto the inward-facing conformation as ATP disso-ciates during extensive washing. Indeed, thiswould be expected, given that the apparent af-finity of ATP binding, as assessed through the

Kim et al., Science 359, 915–919 (2018) 23 February 2018 2 of 5

Fig. 2. Two symmetrical ATPase sites. (A) TheQ loops (blue) in the NBD dimer are positionedto interact with Mg2+ and ATP (magenta andyellow) and the TMDs through the two intracellularhelices IH2 and 4 (orange). The highly conservedglutamine residues (Q475 and Q1118) in the Q loopsare indicated. (B) Molecular interactions at eachATPase site, together with the density of Mg2+-ATP(green mesh). Y, tyrosine; R, arginine. (C) Zoom-inviews of the Q loops. Interactions between theQ loop glutamine and Mg2+ and ATP are indicatedby dashed lines.

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ATP dependence of hydrolysis, is in the range of0.2 to 0.7 mM (Fig. 1C) (20, 24, 39–41).The conserved glutamine in the Q loop is one

of the most studied residues in Pgp (Q475 inNBD1 and Q1118 in NBD2). Mutating the Q loopin mouse and human Pgp reduced—and, in thecase of double mutants, abolished—both ATPaseand drug-transporter activity (42–45 ). Puzzlinglythough, linking the two TMDs together with aflexible cross-linker restores the ATPase activityof the double glutamine-to-alanine (Q-to-A) mu-tant (45 ). It is debated whether the function ofthe Q loop is to coordinate the Mg2+ ion for ATPhydrolysis, to communicate the chemistry atthe ATPase sites to conformational changes inthe TMDs, or to facilitate formation of the NBD-closed conformation (42–45 ). The structure ofPgp indicates that the Q loop is critically posi-tioned to perform all three tasks (Fig. 2, A and

C). In both NBDs, the Q loop is part of the inter-face between NBD and TMD, interacting withthe coupling intracellular helixes IH2 and 4 (fig.S4C). They also contribute to the closed NBD-dimer interface by directly contacting the op-posite NBD. Furthermore, Q475 and Q1118 reachinto the ATP binding sites, each coordinating aMg2+ ion and the g-phosphate of ATP that makesvan der Waals contacts with the ABC signaturemotif of the opposite NBD (Fig. 2, A and C).Substitutions of the Q loop glutamine may dis-turb the precise geometry of the ATPase sitenecessary for hydrolysis and destabilize the NBDdimer. Bringing the two TMDs closer could com-pensate for the latter effect, thus explaining whythe cross-linker restores the activity of the doubleQ-to-A mutant (45).The drug-binding cavity observed in the inward-

facing structures is altered completely upon

NBDdimerization (Fig. 3, A and B): Only a smallopening is observed at the extracellular side ofthe membrane. Drug-binding residues, distrib-uted on the surface of the inward-facing cavity,are reoriented toward the extracellular space.Although vinblastine was included in the sam-ple at a concentration fivefold higher than theapparent Km (Fig. 1B), no density correspond-ing to vinblastine was observed. This observationis consistent with biochemical data demonstrat-ing that Pgp has a lower drug affinity in the pres-ence of ATP (46–50). Recent experiments carriedout in native membranes show that bindingof the nonhydrolyzable ATP analog adenylyl-imidodiphosphate is sufficient to induce drugrelease (51). Thus, it is likely that the cryo-EMstructure represents a posttranslocation stateinwhich the substrate has already been releasedto the extracellular side of the membrane.The extracellular segments of the TMDs have

relatively less-defined EM densities and higherB factors (fig. S3 and Fig. 3C), indicating thatthese regions are flexible. Independently, DEERmeasurements of the outward-facing state showedthat distances measured between pairs of res-idues in the extracellular side of the TMDs weredistributed over a broad range instead of a sin-gle population, indicating the presence of multi-ple conformations (25). As Pgp is a promiscuoustransporter interacting with a large variety ofcompounds, the intrinsic flexibility of the extra-cellular segments enables prompt closure of theoutward-facing cavity, thereby preventing reentryof the substrate or other compounds into thetranslocation pathway.As illustrated in Fig. 4A, the structures of var-

ious Pgp orthologs determined in the absenceof ATP have a similar inward-facing architec-ture. Transition from the inward- to the outward-facing conformation involves global movementof the two halves of the molecule as well as ex-tensive local rearrangements of TM helices(Fig. 4). The two “crossing” helices in each TMD(TM4 and 5 in TMD1 and TM10 and 11 in TMD2)pivot inward, bringing the NBDs closer to eachother (Fig. 4A). In addition, the extracellularregions of TM7 and 8 pull away from TM9 to12, resulting in an outward-facing configuration(Fig. 4A).A prominent local conformational change oc-

curs in helices flanking the lateral opening to themembrane inner leaflet (Fig. 4B). In the inward-facing conformation, these helices are interruptedby flexible loops (Fig. 4B) (30, 31). Mutations ofC. merolae Pgp (cmPgp) that changed a disor-dered loop in TM4 into a continuous helix alsodiminished transport activity (31), underscoringthe functional importance of the helix breakers.In the crystal structure of C. elegans Pgp, twomaltoside detergent molecules are observed atthe lateral opening (Fig. 4B), suggesting that theseloops function as binding sites for substrates orflexible hinges to gate the drug-translocationpathway (31). In the outward-facing conforma-tion, however, both TM4 and 10 are continuoushelices (Fig. 4, A and B). The continuity of thesehelices is important to completely close the

Kim et al., Science 359, 915–919 (2018) 23 February 2018 3 of 5

Fig. 3. The translocation pathway. (A) The inward-facing [Protein Data Bank (PDB) 4F4C] and(B) the outward-facing (this study) conformations. The TM cavity is shown as a blue mesh.Drug-interacting residues, which were protected from inhibition in the presence of drugs, are shownas magenta spheres. (C) B-factor distribution of human Pgp is shown by using different colors.The extracellular region is relatively more flexible. Numbers identify the TM helices.

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intracellular gate upon NBD dimerization, avoid-ing potential leakage in the outward-facing state.Another observation revealed in our analy-

sis is that the NBD-TMD interfaces are largelyunchanged during the switch from the inward-to outward-facing conformation (Fig. 4C). TheNBD-TMD interface is important in transmittingconformational changes associated with ATPhydrolysis to substrate translocation. Multiplestructures of the maltose importer show that thecouplinghelix rotates relative to theNBDsduring atransport cycle (52). By contrast, the equivalentinterfaces in Pgp (NBD1-IH4 and NBD2-IH2) aremaintained in the two different conformations(Fig. 4C). Only small movements are observed forIH1 and 3, as these regions engage additionalcontacts with the opposite NBD upon dimeri-zation. Similarly, analysis of different inward-facing Pgp structures shows that the NBD-TMDinterface is largely unchanged (28), suggestingthat theNBDand the intracellular helical region ofthe TMDmove as one concerted rigid body in thetransport cycle.The structure of the human Pgp determined

in this study is stabilized by mutations that pre-vent ATP hydrolysis, which suggests it likelycorresponds to a state that occurs after NBDdimerization but before ATP has been hydro-lyzed. The observations that the drug-bindingsite is collapsed and the substrate is absent inthis conformation suggest that substrate releaseoccurs before ATP hydrolysis. Incorporating thisinformation into the large body of data on Pgp,we hypothesize a model in which the unusuallydynamic nature of Pgp enables unidirectionaltransport of a broad range of substrates (fig. S5).In a transport cycle, Pgp isomerizes between twobasic states: an inward-facing state where NBDsare separated and an outward-facing state whereNBDs are dimerized. In the inward-facing state,the two halves of Pgp pivot to modulate the drug-binding cavity, thereby enabling Pgp to recruitsubstrates of different sizes. Local conformation-al flexibility around the lateral gate further fa-cilitates substrate entry from the inner leafletof the membrane. Upon ATP binding, the trans-porter isomerizes to the outward-facing state,and the drug-binding site is rearranged so thatits affinity for the substrate is lower than whenthe transporter is in the inward-facing state.The outer leaflet regions of the TM helices areflexible, allowing substrate release and subse-quent closure of the translocation pathway. Inthe outward-facing conformation, two ATP mol-ecules are bound to stabilize the NBD dimer. Sub-sequent ATP hydrolysis, which may occur atonly one of the two catalytic sites stochastically(i.e., if two ATP molecules are required to holdthe NBDs together), resets the transporter tothe inward-facing state.

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Fig. 4. Conformational changes of Pgp from inward- to outward-facing states. (A) Structures of tworepresentative inward-facing Pgp and the outward-facing Pgp.The flexible regions in TM4 and 10 are coloredin red. Rotations of TM4 and 5 (orange) and TM10 and 11 (blue) necessary for the transitions areindicated by black arrows. (B) Closure of the lateral opening and conformational changes of the flankinghelices. Surface presentation of the TM region showing that loops in TM4 of cmPgp and TM10 of C. elegansPgp are continuous helices in the outward-facing structure of human Pgp. Detergent molecules are shownas green and red sticks. (C) Few changes are observed at the TMD-NBD interface. Structures of the inward-facing mouse Pgp (PDB 5KPD) and the outward-facing human Pgp are superpositioned with respect tothe NBDs. The two structures are differentiated by the color shading (inward-facing, lighter; outward-facing,darker). The surface clefts into which the intracellular helices (IH2 and 4) docked into are outlined in gray.

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ACKNOWLEDGMENTS

We thank M. Ebrahim and J. Sotiris at Rockefeller’s Evelyn GrussLipper Cryo–Electron Microscopy Resource Center and Z. Yu,C. Hong, and R. Huang at the Janelia Farms Howard Hughes MedicalInstitute cryo-EM facility for assistance in data collection. Wealso thank A. Senior for discussion and comments, S. McCarry forediting this manuscript, and M. Oldham for helping with figures.Funding: J.C. is an investigator in the Howard Hughes MedicalInstitute. Author contributions: Y.K. performed all experiments.Y.K. and J.C. analyzed the structure and wrote the manuscript.Competing interests: The authors declare no competing interests.Data and materials availability: The cryo-EM density map and theatomic coordinates have been deposited in the EMDataBank andthe Protein Data Bank under accession codes EMD-7325 and6C0V, respectively. The expression vector, pEG BacMam, wasprovided by E. Gouaux under a material transfer agreement with theOregon Health and Science University. All other data are available inthe manuscript or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/359/6378/915/suppl/DC1Materials and MethodsFigs. S1 to S5Table S1References (53–70)

12 December 2017; accepted 16 January 2018Published online 25 January 201810.1126/science.aar7389

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Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformationYoungjin Kim and Jue Chen

originally published online January 25, 2018DOI: 10.1126/science.aar7389 (6378), 915-919.359Science 

, this issue p. 915Sciencepromotes the transition to the outward-facing conformation and substrate release.of the cell, but compressed, and no substrate is bound. This suggests that ATP binding, rather than ATP hydrolysis,

outsidetwo nucleotide-binding domains. The substrate-binding site, located in the transmembrane domain, is open to the electron microscopy structure of human PgP in an outward-facing conformation. Two ATP molecules are bound between

−the inward-facing conformation have been determined for mouse, yeast, and algal PgP. Kim and Chen present the cryo substrates out of the cell. Many of its substrates are drugs, so it plays an important role in drug resistance. Structures in

Permeability glycoprotein (PgP) uses the energy from adenosine triphosphate (ATP) hydrolysis to transportA path to multidrug resistance

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