MEMBRANE TRANSPORT

Mechanism of allosteric modulationof P-glycoprotein by transportsubstrates and inhibitorsReza Dastvan1, Smriti Mishra1, Yelena B. Peskova2,Robert K. Nakamoto2, Hassane S. Mchaourab1*

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporterP-glycoprotein plays a central role in clearance of xenobiotics in humans and isimplicated in cancer resistance to chemotherapy. We used double electron electronresonance spectroscopy to uncover the basis of stimulation of P-glycoproteinadenosine 5′-triphosphate (ATP) hydrolysis by multiple substrates and illuminatehow substrates and inhibitors differentially affect its transport function. Our resultsreveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilizationof a high-energy, post-ATP hydrolysis state characterized by structurally asymmetricnucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycleand by high-affinity inhibitors in favor of structurally symmetric nucleotide bindingsites. Together with previous data, our findings lead to a general model of substrateand inhibitor coupling to P-glycoprotein.

Efficient substrate extrusion by ATP-bindingcassette (ABC) efflux transporters, includ-ing the mammalian P-glycoprotein (Pgp),entails the transduction of adenosine 5′-triphosphate (ATP) energy, harvested in

nucleotide-binding domains (NBDs), to proteinconformational motion in transmembrane do-mains (TMDs) (1–11). Transport models for Pgphave emerged from an expanding database ofstructures (12–14), the elucidation of its substrate-coupled conformational dynamics (8), and bio-chemical studies of ATP turnover (15–18).Convincing evidence supports a two-strokemodelwith alternating ATP hydrolysis at the twonucleotide-binding sites (NBSs) (16, 18, 19).Investigation of the substrate-coupled confor-mational cycle of Pgp by double electron elec-tron resonance (DEER) spectroscopy (20–22)bolstered this model by uncovering a vanadate-trapped high-energy posthydrolysis state (HES),previously referred to as the transition state ofATP hydrolysis (23, 24), that results from hydrol-ysis of ATP molecules in structurally and cat-alytically asymmetric NBSs (8). Concomitantwith the formation of the HES, the transportersamples occluded (OO) and outward-facing (OF)conformations, suggesting that the power strokefor transition between inward-facing (IF) andOFstates requires ATP hydrolysis. By contrast, arecent cryo–electron microscopy (cryo-EM) struc-ture of an ATP-bound Pgp mutant in an OFconformation (25) motivated a transport modelwherein substrate extrusion precedes initiationof ATP hydrolysis. Themutant was impaired for

ATP hydrolysis by glutamine substitution of twocatalytic glutamate residues (Fig. 1A), substitu-tions that were previously demonstrated toabrogate NBS asymmetry and stabilize the OFconformation by ATP binding (8).Resolving the discrepancy between the two

models requires elucidation of how substratebinding in the TMD is allosterically coupled toATP hydrolysis. For this purpose, we comparedPgp’s conformational cycles in the absence (basalcycle) and presence of the substrate verapamil(Ver), which accelerates ATP turnover (stimu-lated cycle). Distance distributions for selectedspin-label pairs that were previously shown tofingerprint the IF-to-OF transition (8) were mea-sured in lipid nanodiscs (Fig. 1) (26) and inmixed-detergent/lipid micelles (figs. S1 and S2).For both basal and stimulated cycles (Fig. 1B), weobserved a pattern of distance changes betweenligand-free (apo) Pgp (black traces in Fig. 1) andthe HES (trapped by vanadate after ATP hydrol-ysis, ADP-Vi, red traces in Fig. 1) consistent withthe model of alternating access described pre-viously (8). Assembly of the NBD catalytic dimerin the HES (e.g., residue pair 607–1252), whichbrings together the Walker and ABC signaturemotifs to form the NBSs, is coupled to the ho-mogeneous closure of the intracellular TMD,manifested by almost complete shift of the dis-tributions to shorter distances relative to thecorresponding IF, apo-Pgp distributions. At theextracellular side, the TMD undergoes an open-ing movement as evidenced by distinct longer-distance components. However, independent ofsubstrate binding, a substantial fraction of apo-like distances persists in the extracellular distribu-tions in contrast to the intracellular side of theTMD, which suggests the population of an OOconformation (extracellularly closed) in additionto an OF conformation (extracellularly open).

For most spin-label pairs, we observed mi-nor differences (≅10%) between the basal andsubstrate-coupled HES distance distributions(see fig. S1), which is consistent with efficientVi trapping and similar overall conformation.Substantial changes upon substrate binding werelimited to the distributions of spin-label pairsmonitoring the conserved A-loops (27), whichare located in the NBSs and directly pack againstthe adenosine of bound ATP through a con-served tyrosine (Figs. 2A, 1C). A distinct short-distance component (~67%) is evident in thesubstrate-coupled HES (Fig. 1C) at the NBS2A-loop (511–1043), but not at the equivalent pairat the NBS1 A-loop (400–1156). We previouslyinterpreted the structural asymmetry betweenA-loops as a signature of high-affinity occlusionof ATP in NBS2 and its hydrolysis in NBS1 (8),which parallels the intrinsic catalytic asymmetryof substrate-coupled Pgp (16, 18). By contrast,for substrate-free HES, the short-distance com-ponent at NBS2 is a minor population (~25%)in nanodiscs (Fig. 1C) and negligible (~7%) inmixed micelles (Fig. 1D). Thus, the basal andstimulated cycles differ by the heterogeneityand asymmetry of the A-loops in otherwisestructurally similar OO and OF conformations.Whereas DEER distance distributions in the

NBD and the intracellular TMD were consistentwith the cryo-EM structure of the double E/Qmutant, A-loop asymmetry was not observed inthis presumed OF structure (Fig. 1 and fig. S2),thus predicting similar short-distance compo-nents for pairs monitoring the A-loops. Thisdeviation is rationalized by the finding thatA-loop asymmetry can be abrogated by thedouble E/Qmutation (8) and reversed by a singleE/Q mutation (fig. S3). Another notable devi-ation is evident on the extracellular side, whichis more closed (apo-like) in the cryo-EM struc-ture relative to the HES (fig. S2). The cryo-EMdensity at the extracellular side was diffuse,suggesting a dynamic region (25), a findingconsonant with the heterogeneous experimentalDEER distributions.To uncover the mechanistic implications of

the asymmetric HES, we determined how a setof substrates, which display different transportefficiency (28) and ATP turnover rates (15) (figs.S4 to S6), modulate A-loop heterogeneity andasymmetry (Fig. 2 and fig. S7). Furthermore,Arrhenius analysis of ATP turnover for a subsetof these substrates demonstrated a commonrate-limiting step (15). Because transport sub-strates can inhibit ATP turnover at high con-centrations, saturating stimulatory substrateconcentrations were selected (determined infigs. S4B and S6 and tables S1 and S2). Wefound that substrates most efficient in stim-ulating ATP turnover (e.g., Ver) induced thelargest population of the short component atNBS2. Indeed, a linear relationship emergedbetween this component’s population, repre-senting the spectroscopic asymmetric HES, andthe natural logarithm of stimulated ATP turn-over rate constant (kcat). Other pairs in the NBDand TMD did not show substantial population

RESEARCH

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1Department of Molecular Physiology and Biophysics,Vanderbilt University, Nashville, TN 37232, USA.2Department of Molecular Physiology and BiologicalPhysics, University of Virginia, Charlottesville,VA 22908, USA.*Corresponding author. Email: hassane.mchaourab@vanderbilt.edu

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changes (i.e., >10%), which exclude the possibil-ity of heterogeneous Vi trapping and thereforelink the allosteric effects of substrate binding tothe conformation of A-loops. Given that Ln (kcat)reflects the activation energy of the hydrolytic tran-sition state, we conclude that substrate-inducedreduction in the activation energy requires sta-bilization of the catalytically and structurally asym-metric HES. Moreover, distinct kcat values andpopulations of HES asymmetry imply differen-tial interaction of the substrates with this state,which has OO and OF conformations. Thus, sub-strate extrusion must occur simultaneously withor subsequent to ATP hydrolysis.In contrast to transport substrates, the Pgp

high-affinity inhibitors zosuquidar and tariquidar(12, 29) (Figs. 2C and 3 and fig. S8) inducedbroader distance distributions at NBS1, suggest-ing the population of an intermediate-distancecomponent in the HES. A similar componentwas observed at NBS2 (black arrows in Fig. 2C),whereas the population of the short-distancecomponent, stabilized by substrates, was sim-ilar to that of the basal cycle for zosuquidar andwas reduced for tariquidar. The intermediate-distance components reveal a distinct, more sym-metric conformation of the A-loops associatedwith high-affinity inhibitor binding.To uncover how high-affinity inhibitors ab-

rogate transport and destabilize the asymmetricHES while stimulating ATP turnover (30), wemapped structural changes in apo-Pgp inducedby inhibitor binding (fig. S8, C and D). Althoughthe distance distributions were heterogeneous,suggesting a conformational equilibrium, a dis-tinct IF conformation was observed wherein theTMD intracellular side and NBDs were closerrelative to apo-Pgp (solid orange traces, blackarrows in Fig. 3 and fig. S8). By contrast, dis-tance distributions were unchanged upon theaddition of excess substrates to apo-Pgp (fig. S8B),indicating a lack of structural changes. Moreover,the IF conformation stabilized by inhibitors per-sisted in the HES (ADP-Vi, dark red traces inFig. 3), suggesting that inhibitors not only re-duce the asymmetry at the A-loops but also im-pair homogeneous closing of the intracellularTMD and the formation of an OF conformation(8). The direction of the distance changes in theinhibitor-stabilized IF agrees with a recent cryo-EM structure of zosuquidar-bound Pgp (Fig. 3)(12), with one distance component overlappingthe distributions predicted from this structure(dashed orange traces in Fig. 3). However, theconformational equilibrium implied by hetero-geneous DEER distributions is likely restrictedin this structure by binding of an extracellularantibody and a disulfide bond introduced at theNBD interface.Our findings can be framed in a model that

describes how inhibitors and substrates dif-ferentially couple to the Pgp transport cycle.The model (Fig. 4) invokes distinct HESs dif-fering by the configuration of the NBSs butotherwise having almost identical OO/OF con-formations. The basal cycle proceeds primarilythrough the NBS-symmetric HES in which both

Dastvan et al., Science 364, 689–692 (2019) 17 May 2019 2 of 4

A

B

C

D

Fig. 2. Stimulation of ATP turnover by substrates entails stabilization of an asymmetric HES.(A) Cytoplasmic view of the NBD dimer in the OF conformation in complex with ATP (red sticks)showing the A-loop (red spheres) spin-label pairs. (B) Distance distributions of the A-loop pairshighlighting the substrate dependence of the short-distance component (arrow) at NBS2. (C) Pgpinhibitors stabilize an HES with a distinct distance component (arrows) relative to substrates.(D) The asymmetric HES population for transport substrates is directly related to the activationenergy of ATP turnover (Ln kcat). Experiments in (B) to (D) were performed in nanodiscs. Distancedistributions were obtained in the trapped (ADP-Vi) HES.

Fig. 1. HESs for basal andsubstrate-coupled cycles differby the conformation of theA-loops. (A) Ribbon representa-tion of the OF Pgp [ProteinData Bank (PDB) code 6C0V]with the N- and C-terminal halvescolored with orange and cyan,respectively, and highlighting thepositions of spin-label pairs aspurple spheres. (B) Distance dis-tributions in the TMDs andNBDs and (C and D) the A-loopsobtained in nucleotide-free Pgp(Apo) and the HES (ADP-Vi) inthe presence and absence of thesubstrate Ver in nanodiscs. Thecorresponding distributions pre-dicted from the cryo-EM OFstructure are shown as dashedlines. (D) Distance distributionsfor NBS2 in mixed micelles areshown for reference. Residue 92is not resolved in the cryo-EMstructure, precluding predictionof distance distributions.

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ATP molecules have been hydrolyzed presum-ably in random order (Fig. 4, step 2). Because Vitrapping of ADP, subsequent to ATP hydrolysis,can only occur in oneNBS at a time (8, 16, 18), thesymmetric HES indicates that the basal cyclehas reduced catalytic asymmetry.Stimulation of ATP hydrolysis by substrates

proceeds through an NBS-asymmetric HES, in

which one ATP molecule is hydrolyzed whereasthe other is occluded with high affinity (Fig. 4,step 4a). We propose that asymmetric occlusionof ATP lowers the enthalpic barrier for hydrolysisand consequently accelerates the overall cycle,which is manifested by a larger kcat. The substrate-dependent asymmetric HES population is con-sistently less than100%, implying that the symmetric

HES is alsopopulated in the substrate-stimulatedcycle. Whereas it is possible that the substrate-coupled cycle involves a transient conformationbound to twoATPmolecules similar to the cryo-EMstructure (25), our findings establish that substrateextrusion cannot proceed until at least one ATP ishydrolyzed. Stabilization of the asymmetric HESfor Pgp reconstituted in nanodiscs compared

Dastvan et al., Science 364, 689–692 (2019) 17 May 2019 3 of 4

Fig. 4. Model of Pgp transport and inhibition. The basalcycle (middle panels, steps 1 and 2) entails conformationalsampling by ATP-bound Pgp to enable NBD dimerizationfollowed by population of a symmetric HES. The substrate-coupled cycle (steps 1, 3a, 4a, and 2) is initiated bysubstrate binding. As previously postulated (8, 16, 18), atransient conformation with one tightly bound ATP mole-cule is likely populated. Hydrolysis of one ATP stabilizes theasymmetric HES (step 4a), which consists of OO/OFconformations. Hydrolysis of the second ATP leads to thesymmetric HES as in the basal cycle (step 2). Pgpinhibition (steps 1, 3b, and 4b) is initiated by stabilization ofan IF conformation in which the extracellular sides ofTMD is apo-like, whereas the intracellular sides of TMDsand NBDs are closer than the apo state. ATP hydrolysisproceeds through a heterogeneous HES. For each inter-mediate, we show cytoplasmic views of the NBDs high-lighting the conserved A-loop tyrosines as purple spheresalong with associated DEER distributions in nanodiscs.

Fig. 3. The high-affinity inhibitor zosuquidarstabilizes a distinct IF conformation ofPgp. (A) Ribbon representation of zosuquidar-bound Pgp cryo-EM structure (PDB code6FN1) highlighting the positions of spin-labelpairs as purple spheres. (B) Distance distri-butions obtained in nanodiscs for apo-Pgp,zosuquidar-bound nucleotide-free Pgp, andthe HES (ADP-Vi). Predicted distributions areshown as dotted lines. Arrows highlight com-ponents on the intracellular side of the TMDand at the A-loops that are either not observedor are minor components in apo-Pgp. The yaxes in Figs. 1 and 3 are identical.

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with mixedmicelles (Fig. 1, C and D) rationalizesthe stimulation of basal ATP hydrolysis (tables S1and S2) (26) in the former and suggests that HESstability is lipid dependent. It also structurallycontextualizes substrate-dependent Gibbs energyanalysis of ATP turnover that postulated a singlerate-limiting conformational step (15), i.e. transitionto theOF conformation, and linked intrinsicallydifferent kcat of substrates to their affinities tothis rate-limiting step as observed here for theasymmetric HES.Inhibitors stabilize an NBS-heterogeneous

HES (Fig. 4, step 4b) distinct from that of thebasal cycle. We suggest that high-affinity inhib-itors accelerate ATP hydrolysis by inducing anIF conformation in which the NBDs are closerrelative to ligand-free Pgp, effectively reducingthe conformational entropy of the IF ensemble.The energy of ATP hydrolysis is not sufficientto homogeneously close the intracellular side inthe HES, as evidenced by persistent apo-likedistance components (Fig. 3B, black arrow inpanel 145–787). Accordingly, transport, requiringisomerization to an OF, is impaired by inhib-itor binding.In addition to illuminating the mechanism of

allostericmodulation of Pgpby transport substratesand inhibitors, our approach of monitoring theconformational equilibrium of Pgp—specificallydirect detection of the distinct HES populations—provides a strategy to identify allosteric mod-

ulators for multidrug ABC exporters in general,thereby accelerating the development of newinhibitors.

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ACKNOWLEDGMENTS

We thank B. Verhalen for helpful discussions and D. P. Claxton andR. A. Stein for critical reading and editing of the manuscript.Funding: This work was supported by National Institutes of Healthgrant U54-GM087519 (to H.S.M. and R.K.N.). Author contributions:H.S.M., R.D., and R.K.N. designed the experimental studies. R.D.and S.M. purified the mutants and reconstituted in nanodiscs. R.D.performed the experiments and analyzed the data. H.S.M. andR.D. wrote the paper. Y.B.P. cloned and expressed the mutants.Competing interests:The authors declare that there are no competinginterests. Data and materials availability: The generated data,including those from the DEER experiments, are available in themanuscript or the supplementary materials.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/364/6441/689/suppl/DC1Materials and MethodsFigs. S1 to S8Tables S1 and S2DEER Data AppendicesReferences (31–36)

2 November 2018; accepted 17 April 201910.1126/science.aav9406

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Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitorsReza Dastvan, Smriti Mishra, Yelena B. Peskova, Robert K. Nakamoto and Hassane S. Mchaourab

DOI: 10.1126/science.aav9406 (6441), 689-692.364Science 

, this issue p. 689ScienceATP-hydrolysis state. By contrast, inhibitors stabilize a symmetric state that impairs transport.

−electron resonance spectroscopy to show that substrates enhance transport by stabilizing an asymmetric post used double electron et al.mechanism, but a full picture requires access to substates in the transport cycle. Dastvan

Structures of P-glycoprotein in the apo state and bound to substrate and inhibitor give insight into the transporthydrolysis to expel chemical substances, including drugs. Inhibiting P-glycoprotein may thus ameliorate drug resistance.

The membrane protein P-glycoprotein protects cells by using energy from adenosine triphosphate (ATP)Transport control

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