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Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation

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Science  23 Feb 2018:
Vol. 359, Issue 6378, pp. 915-919
DOI: 10.1126/science.aar7389

A path to multidrug resistance

Permeability glycoprotein (PgP) uses the energy from adenosine triphosphate (ATP) hydrolysis to transport substrates out of the cell. Many of its substrates are drugs, so it plays an important role in drug resistance. Structures in the inward-facing conformation have been determined for mouse, yeast, and algal PgP. Kim and Chen present the cryo–electron microscopy structure of human PgP in an outward-facing conformation. Two ATP molecules are bound between two nucleotide-binding domains. The substrate-binding site, located in the transmembrane domain, is open to the outside of the cell, but compressed, and no substrate is bound. This suggests that ATP binding, rather than ATP hydrolysis, promotes the transition to the outward-facing conformation and substrate release.

Science, this issue p. 915

Abstract

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-powered conformational changes. Despite decades of effort, only the structures of the inward-facing conformation of P-glycoprotein are available. Here we present the structure of human P-glycoprotein in the outward-facing conformation, determined by cryo–electron microscopy at 3.4-angstrom resolution. The two nucleotide-binding domains form a closed dimer occluding two ATP molecules. The drug-binding cavity observed in the inward-facing structures is reorientated toward the extracellular space and compressed to preclude substrate binding. This observation indicates that ATP binding, not hydrolysis, promotes substrate release. The structure evokes a model in which the dynamic nature of P-glycoprotein enables translocation of a large variety of substrates.

Cancer cells develop resistance to chemically diverse compounds, a phenomenon known as multidrug resistance (MDR). To improve the effectiveness of chemotherapy, many laboratories have searched for mechanisms that account for MDR. In 1973, Keld Danø demonstrated that the reduced drug accumulation in tumor cells was energy dependent (1). In 1976, by labeling cell-surface carbohydrates, Juliano and Ling identified a glycoprotein enriched in colchicine-resistant cells but not in wild-type cells (2). The protein was named the permeability (P)–glycoprotein (Pgp) because it was thought to confer drug resistance by making the cellular membrane less permeable (3). Ten years later, the genes responsible for MDR in human, mouse, and hamster (named MDR genes) were cloned (46), and it was shown that the protein product of the mdr1 gene was indeed Pgp (7).

Pgp is an adenosine triphosphate (ATP)–binding cassette (ABC) transporter, which uses the energy from ATP hydrolysis to pump substrates across the membrane. It contains two transmembrane domains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs) (Fig. 1A). Pgp is expressed in many membrane “barriers” of the body, including the blood-brain barrier, gastrointestinal tract, kidney, liver, ovary, and placenta (2, 811). Thus, the physiological function of Pgp is likely to protect sensitive tissues and the fetus from endogenous and exogenous toxicity (12). More than 300 compounds have been identified as potential substrates of Pgp (13, 14). Drug resistance mediated by Pgp depends on ATP hydrolysis (1517), and the adenosine triphosphatase (ATPase) activity of Pgp is stimulated by the transported drugs (1820). Vanadate (Vi) trapping and photocleavage experiments showed that Pgp contains two active ATPase sites, but only one ATP is hydrolyzed at a time (21).

Fig. 1 Characterization of recombinant human Pgp.

(A) Topology diagram of Pgp. Residues not resolved in the structure are shown as dashed lines. OUT, outer cell membrane; IN, inner cell membrane. (B) Vinblastine stimulates the ATPase activity of the wild-type (WT) protein but not the E556Q/E1201Q mutant (EQ). Data points represent the means ± standard deviation of three to nine measurements at 29°C. By nonlinear regression of the Michaelis-Menten equation, WT Pgp in n-dodecyl-β-d-maltopyranoside (DDM) and cholesteryl hemisuccinate (CHS) solution has a Km of 30 ± 3 μM 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 of 100 μM vinblastine, the WT protein has a Km of 0.179 ± 0.05 mM for ATP and a maximal ATPase activity of 260 ± 16 nmol/mg/min. (D) The overall structure 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 and NBD2) 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).

The molecular structures of Pgp in a transport cycle have been investigated by using a variety of methods, including antibody binding (22), tryptophan fluorescence (23), luminescence (24), double electron-electron resonance (DEER) (25), electron microscopy (EM) (26, 27), and x-ray crystallography (2831). Although the crystal structures of Pgp have been determined for the mouse (28, 29), Caenorhabditis elegans (30), and Cyanidioschyzon merolae (31) orthologs, all of these structures exhibit a similar conformation, in which the two NBDs are separated from each other and the translocation pathway is accessible from inside the cell (i.e., the inward-facing conformation). To reveal conformational changes that enable substrate translocation, we pursued the structure of human Pgp in an ATP-bound, outward-facing conformation.

Pgp contains two active ATPase sites, each comprising the Walker A and Walker B motifs of one NBD and the Leu-Ser-Gly-Gly-Gln (LSGGQ) motif of the other NBD. At each ATPase site, a highly conserved glutamate residue acts as the catalytic base for ATP hydrolysis. Mutating either catalytic glutamate severely reduces ATPase activity; concurrent mutations at both positions trap Pgp in an ATP-occluded form, likely with a closed-NBD dimer (3234). On the basis of these data, we generated a mutant of Pgp in which both catalytic glutamates (abbreviated E, at residue positions 556 and 1201) were replaced by glutamines (E556Q/E1201Q). Compared to the wild-type (WT) protein, whose ATPase activity increases as a function of ATP concentration and is stimulated by the substrate vinblastine, the E556Q/E1201Q mutant remains inactive over a large range of ATP and vinblastine concentrations (Fig. 1, B and C).

For cryo-EM studies, the mutant protein was incubated with 150 μM vinblastine and 10 mM Mg2+-ATP to promote ATP occlusion (3234). A cryo-EM data set consisting of ~1 million particles was collected and analyzed for structural homogeneity. Three-dimensional (3D) classification using Relion (35) showed that most of the particles exhibited an NBD-dimerized conformation (fig. S1). Refinement of the best class produced a map at 3.4-Å resolution (figs. S1 and S2), enabling us to build a nearly complete model of Pgp except for the two termini and residues 81 to 104 and 631 to 694, which form an extracellular loop and intracellular linker, respectively (fig. S3).

The overall structure of human Pgp is substantially different from the previously determined inward-facing conformations (Fig. 1D). Instead of enclosing an internal cavity, the transmembrane (TM) helices pack closely in the membrane inner leaflet. The two cytosolic NBDs make extensive contacts with each other, forming the typical “head-to-tail” dimer characteristic of ABC transporters (Fig. 2A). Two ATP molecules are bound at the dimer interface, interacting with the Walker A motif of one NBD and the ABC signature motif of the other NBD (Figs. 1D and 2). This structure, largely consistent with what is expected of an outward-facing conformation, also reveals several unanticipated features that advance our understanding of the transport cycle.

Fig. 2 Two symmetrical ATPase sites.

(A) The Q loops (blue) in the NBD dimer are positioned to interact with Mg2+ and ATP (magenta and yellow) and the TMDs through the two intracellular helices IH2 and 4 (orange). The highly conserved glutamine residues (Q475 and Q1118) in the Q loops are indicated. (B) Molecular interactions at each ATPase site, together with the density of Mg2+-ATP (green mesh). Y, tyrosine; R, arginine. (C) Zoom-in views of the Q loops. Interactions between the Q loop glutamine and Mg2+ and ATP are indicated by dashed lines.

Biochemical studies have shown that mouse Pgp containing alanine substitutions of both catalytic glutamates occludes maximally a single ATP (32, 36). Nucleotide trapping by Vi or beryllium fluoride also occurs at only one of the two catalytic sites (37, 38). This suggests structural asymmetry of the two ATPase sites in the outward-facing conformation. However, the structures of the two NBDs are nearly identical (superposition of all 246 α carbons in the NBDs yields a root mean square deviation of 0.5 Å) (fig. S4A). Furthermore, the cryo-EM densities for both ATPs are equally strong, and the ATP molecules make very similar contacts with nearby residues (Fig. 2B and fig. S4B).

The discrepancy between the structural and biochemical data can be explained by the different experimental conditions. The cryo-EM grids were prepared with saturating concentrations of ATP (10 mM); in the biochemical study, the stoichiometry values were determined after affinity chromatography in an ATP-free buffer (32, 36). Cryo-EM 3D classification analysis shows that, in the presence of 10 mM ATP, about 5% of particles reside in the inward-facing (non–ATP-occluded) conformation (fig. S1). Two other independent EM studies at low resolution have also shown that only a fraction of Pgp molecules could be trapped in the outward-facing conformation by nonhydrolyzable ATP analogs or ATP with Vi (26, 27). Thus, it is possible that, in biochemical studies, a large fraction of the molecules convert to the inward-facing conformation as ATP dissociates during extensive washing. Indeed, this would be expected, given that the apparent affinity of ATP binding, as assessed through the ATP dependence of hydrolysis, is in the range of 0.2 to 0.7 mM (Fig. 1C) (20, 24, 3941).

The conserved glutamine in the Q loop is one of the most studied residues in Pgp (Q475 in NBD1 and Q1118 in NBD2). Mutating the Q loop in mouse and human Pgp reduced—and, in the case of double mutants, abolished—both ATPase and drug-transporter activity (4245). Puzzlingly though, linking the two TMDs together with a flexible cross-linker restores the ATPase activity of the double glutamine-to-alanine (Q-to-A) mutant (45). It is debated whether the function of the Q loop is to coordinate the Mg2+ ion for ATP hydrolysis, to communicate the chemistry at the ATPase sites to conformational changes in the TMDs, or to facilitate formation of the NBD-closed conformation (4245). The structure of Pgp indicates that the Q loop is critically positioned to perform all three tasks (Fig. 2, A and C). In both NBDs, the Q loop is part of the interface between NBD and TMD, interacting with the coupling intracellular helixes IH2 and 4 (fig. S4C). They also contribute to the closed NBD-dimer interface by directly contacting the opposite NBD. Furthermore, Q475 and Q1118 reach into the ATP binding sites, each coordinating a Mg2+ ion and the γ-phosphate of ATP that makes van der Waals contacts with the ABC signature motif of the opposite NBD (Fig. 2, A and C). Substitutions of the Q loop glutamine may disturb the precise geometry of the ATPase site necessary for hydrolysis and destabilize the NBD dimer. Bringing the two TMDs closer could compensate for the latter effect, thus explaining why the cross-linker restores the activity of the double Q-to-A mutant (45).

The drug-binding cavity observed in the inward-facing structures is altered completely upon NBD dimerization (Fig. 3, A and B): Only a small opening is observed at the extracellular side of the membrane. Drug-binding residues, distributed on the surface of the inward-facing cavity, are reoriented toward the extracellular space. Although vinblastine was included in the sample at a concentration fivefold higher than the apparent Km (Fig. 1B), no density corresponding to vinblastine was observed. This observation is consistent with biochemical data demonstrating that Pgp has a lower drug affinity in the presence of ATP (4650). Recent experiments carried out in native membranes show that binding of the nonhydrolyzable ATP analog adenylyl-imidodiphosphate is sufficient to induce drug release (51). Thus, it is likely that the cryo-EM structure represents a posttranslocation state in which the substrate has already been released to the extracellular side of the membrane.

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 shown as 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.

The extracellular segments of the TMDs have relatively less-defined EM densities and higher B factors (fig. S3 and Fig. 3C), indicating that these regions are flexible. Independently, DEER measurements of the outward-facing state showed that distances measured between pairs of residues in the extracellular side of the TMDs were distributed over a broad range instead of a single population, indicating the presence of multiple conformations (25). As Pgp is a promiscuous transporter interacting with a large variety of compounds, the intrinsic flexibility of the extracellular segments enables prompt closure of the outward-facing cavity, thereby preventing reentry of the substrate or other compounds into the translocation pathway.

As illustrated in Fig. 4A, the structures of various Pgp orthologs determined in the absence of ATP have a similar inward-facing architecture. Transition from the inward- to the outward-facing conformation involves global movement of the two halves of the molecule as well as extensive 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 each other (Fig. 4A). In addition, the extracellular regions of TM7 and 8 pull away from TM9 to 12, resulting in an outward-facing configuration (Fig. 4A).

Fig. 4 Conformational changes of Pgp from inward- to outward-facing states.

(A) Structures of two representative inward-facing Pgp and the outward-facing Pgp. The flexible regions in TM4 and 10 are colored in red. Rotations of TM4 and 5 (orange) and TM10 and 11 (blue) necessary for the transitions are indicated by black arrows. (B) Closure of the lateral opening and conformational changes of the flanking helices. Surface presentation of the TM region showing that loops in TM4 of cmPgp and TM10 of C. elegans Pgp are continuous helices in the outward-facing structure of human Pgp. Detergent molecules are shown as 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 to the 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.

A prominent local conformational change occurs in helices flanking the lateral opening to the membrane inner leaflet (Fig. 4B). In the inward-facing conformation, these helices are interrupted by flexible loops (Fig. 4B) (30, 31). Mutations of C. merolae Pgp (cmPgp) that changed a disordered loop in TM4 into a continuous helix also diminished transport activity (31), underscoring the functional importance of the helix breakers. In the crystal structure of C. elegans Pgp, two maltoside detergent molecules are observed at the lateral opening (Fig. 4B), suggesting that these loops function as binding sites for substrates or flexible hinges to gate the drug-translocation pathway (31). In the outward-facing conformation, however, both TM4 and 10 are continuous helices (Fig. 4, A and B). The continuity of these helices is important to completely close the intracellular gate upon NBD dimerization, avoiding potential leakage in the outward-facing state.

Another observation revealed in our analysis is that the NBD-TMD interfaces are largely unchanged during the switch from the inward- to outward-facing conformation (Fig. 4C). The NBD-TMD interface is important in transmitting conformational changes associated with ATP hydrolysis to substrate translocation. Multiple structures of the maltose importer show that the coupling helix rotates relative to the NBDs during a transport cycle (52). By contrast, the equivalent interfaces in Pgp (NBD1-IH4 and NBD2-IH2) are maintained in the two different conformations (Fig. 4C). Only small movements are observed for IH1 and 3, as these regions engage additional contacts with the opposite NBD upon dimerization. Similarly, analysis of different inward-facing Pgp structures shows that the NBD-TMD interface is largely unchanged (28), suggesting that the NBD and the intracellular helical region of the TMD move as one concerted rigid body in the transport cycle.

The structure of the human Pgp determined in this study is stabilized by mutations that prevent ATP hydrolysis, which suggests it likely corresponds to a state that occurs after NBD dimerization but before ATP has been hydrolyzed. The observations that the drug-binding site is collapsed and the substrate is absent in this conformation suggest that substrate release occurs before ATP hydrolysis. Incorporating this information into the large body of data on Pgp, we hypothesize a model in which the unusually dynamic nature of Pgp enables unidirectional transport of a broad range of substrates (fig. S5). In a transport cycle, Pgp isomerizes between two basic states: an inward-facing state where NBDs are separated and an outward-facing state where NBDs are dimerized. In the inward-facing state, the two halves of Pgp pivot to modulate the drug-binding cavity, thereby enabling Pgp to recruit substrates of different sizes. Local conformational flexibility around the lateral gate further facilitates substrate entry from the inner leaflet of the membrane. Upon ATP binding, the transporter isomerizes to the outward-facing state, and the drug-binding site is rearranged so that its affinity for the substrate is lower than when the transporter is in the inward-facing state. The outer leaflet regions of the TM helices are flexible, allowing substrate release and subsequent closure of the translocation pathway. In the outward-facing conformation, two ATP molecules are bound to stabilize the NBD dimer. Subsequent ATP hydrolysis, which may occur at only one of the two catalytic sites stochastically (i.e., if two ATP molecules are required to hold the NBDs together), resets the transporter to the inward-facing state.

Supplementary Materials

www.sciencemag.org/content/359/6378/915/suppl/DC1

Materials and Methods

Figs. S1 to S5

Table S1

References (5370)

References and Notes

Acknowledgments: We thank M. Ebrahim and J. Sotiris at Rockefeller’s Evelyn Gruss Lipper Cryo–Electron Microscopy Resource Center and Z. Yu, C. Hong, and R. Huang at the Janelia Farms Howard Hughes Medical Institute cryo-EM facility for assistance in data collection. We also thank A. Senior for discussion and comments, S. McCarry for editing this manuscript, and M. Oldham for helping with figures. Funding: J.C. is an investigator in the Howard Hughes Medical Institute. 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 the atomic coordinates have been deposited in the EMDataBank and the Protein Data Bank under accession codes EMD-7325 and 6C0V, respectively. The expression vector, pEG BacMam, was provided by E. Gouaux under a material transfer agreement with the Oregon Health and Science University. All other data are available in the manuscript or the supplementary materials.
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