...catalytic site is sufficient to promote transport in P...

来自 : 发布时间：2025-03-31

AbstractP-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis. IntroductionP-glycoprotein (Pgp) is a primary active membrane transporter of the ABC (ATP Binding Cassette) protein superfamily. It is the first human ABC transporter discovered to be responsible for the increased efflux of chemotherapeutics from multidrug resistant cancer cells1,2. Pgp is referred to as a 鈥渉ydrophobic vacuum cleaner鈥? because it is believed to extract its substrates directly from the inner leaflet of the plasma membrane3. This molecular mechanism of action provides an incredibly efficient efflux of a vast array of hydrophobic drugs, ensuring the survival of cancer cells despite toxic chemotherapy (for reviews see4,5).Based on biochemical experiments and the commonality of various mammalian and bacterial ABC transporter structures it is generally believed that the fundamental molecular mechanism of substrate transport is shared among ABC transporters6,7,8. From bacteria to humans, ABC transporters are composed of at least two membrane-embedded transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs). The TMDs define the substrate binding sites and the translocation pathway and the NBDs bind and hydrolyze ATP. The TMDs are connected to the NBDs by intra-cytosolic loop (ICL) domains, which transfer signals to coordinate ATP binding and hydrolysis with substrate transport7. The NBDs contain several evolutionarily conserved sequences, including the Walker A and B motifs that are commonly found in nucleotide-binding proteins9 and the so-called signature sequence (C-loop or LSGGQ motif), which is unique to the ABC protein family10. The conserved motifs form two composite catalytic sites in which the ATP molecules are sandwiched between the Walker A and Walker B motifs of one NBD and the signature sequence of the contralateral NBD11.Crystal structures of full-length ABC transporters have revealed two major conformations: in the absence of nucleotides, the NBD dimers are dissociated and the TMDs adopt an inward-facing conformation6,12. Nucleotide binding to the dissociated NBDs13 induces formation of two composite catalytic sites at the interface of a tight head-to-tail NBD1/NBD2 heterodimer, glued together by the two ATP molecules. Concomitantly with NBD dimer formation the TMDs flip into an outward-facing conformation7,8. Only one of two gates is open at any time: in the inward-facing conformation the cytoplasmic gate of the translocation pathway is open, whereas the extracellular gate is closed. Conversely, in the outward-facing conformation the cytoplasmic gate is closed and the transported drug is free to dissociate to the extracellular compartment. The above conformational transitions are accompanied by the switch of the affinity of the substrate binding sites from high- to low-affinity to ensure substrate transport against the concentration gradient14,15.Notably, the recently solved structure of the antibacterial peptide ABC transporter McjD is occluded on both sides of the membrane. This conformation, termed nucleotide-bound outward occluded, probably represents a transition intermediate between the outward-open and inward-open TMD conformations of ABC exporters16. Repeated formation and disruption of the NBD dimer is generally agreed to involve the cooperative, ATP-dependent interaction of the NBDs, but because the resolved structures do not represent all phases of the transport cycle the exact sequence of events is unknown. While the major principles of the ATP-dependent transport mechanism are shared among ABC transporters, details of the coupling of the TMD transitions to the ATPase cycle may differ in different subclasses of ABC exporters17.A spate of biochemical data supports that the molecular mechanism of Pgp follows the above alternating access scheme (reviewed in18,19). Still, despite the availability of the mouse6,20 and the C. elegans12 Pgp structures, the molecular mechanisms linking ATP binding and/or hydrolysis to the association and dissociation of NBDs and to conformational changes occurring in the TMDs are not fully understood. The catalytic cycle is driven by ATP hydrolysis, which may happen at either of the two functionally symmetrical composite nucleotide binding sites. How ATP hydrolysis is coordinated between the two NBDs and whether Pgp hydrolyses one or two (or more) ATP molecules per each transported substrate are not known. Experiments attempting to explain the sequence of molecular events that lead to the ATP-dependent conformational switch and the change of substrate binding affinity have produced competing models21.Conformational transitions in Pgp that occur during transport-associated ATP hydrolysis can be detected by differential immunoreactivity to the monoclonal antibody UIC222. The complex epitope of UIC2 consists of multiple extracellular loops of Pgp23,24, which undergo conformational changes when the TMDs rearrange to bind and release the transported substrate. It was shown that UIC2 binding to Pgp is increased in the presence of transported substrates, ATP-depleting agents, or by mutational inactivation of both nucleotide-binding domains22, suggesting that UIC2-detectable conformational transitions are driven by binding and unbinding of nucleotides25. In the present study we correlated UIC2-reactivity with drug binding affinity for wild-type and mutant Pgp variants in permeabilized cells to elucidate the link between ATP binding, hydrolysis and the conformational rearrangements responsible for switching the affinity of the substrate binding sites during substrate transport.ResultsNucleotide binding switches Pgp into the outward facing conformationGentle permeabilization of cells allows selective modulation of the intracellular milieu while preserving membrane integrity and the function of transmembrane transporters. This experimental setup has been used before to synchronize Pgp molecules in an ATP-free, high UIC2-affinity conformation25. Similarly to the results obtained by Druley and co-workers25, replenishing the cells with ATP/Mg2+ shifted Pgp into a low UIC2-affinity conformation in a concentration dependent manner (Fig. 1a). The apparent affinity of ATP for competing UIC2 labeling (KA, Supplementary Table S1) was found to be comparable with reported KM values for ATP hydrolysis26,27 as well as with our results obtained in membrane samples prepared from Pgp+ NIH 3T3 cells (Supplementary Fig. S1).Figure 1UIC2 mAb binding to Staphylococcus alpha toxin permeabilized NIH 3T3 cells expressing wild-type Pgp in the presence of ATP/Mg2+ (a) ATP in the absence of Mg2+ (b) AMP-PNP/Mg2+ (c) or ATP/Mg2+ at 4鈥壜癈 (d). Permeabilized cells were treated with nucleotides at different concentrations for 20鈥塵in and then labeled with UIC2-A647 for another 30鈥塵in (a鈥揷) or 40鈥塵in (d) without washing out the nucleotides. In case of Vi treatment un-trapped nucleotides were washed out in the presence of Vi. UIC2-A647 fluorescence intensities were plotted as a function of nucleotide concentration and fitted with a four parameter Hill function to determine the apparent affinities of nucleotide binding (KA) and Hill slope values (see Materials and Methods).Full size imageAMP-PNP, a non-hydrolysable ATP analogue also induced a conformation change in the transmembrane domains resulting in a concentration dependent decrease in UIC2 reactivity (Fig. 1c). When ATP was added in the absence of Mg2+ (ATP鈥?鈥塃DTA; Fig. 1b) or on ice (Fig. 1d), i.e., under conditions that prevent ATP hydrolysis, the conformational change driving Pgp into a UIC2-dim state was observed at significantly higher nucleotide concentrations (Supplementary Table S1).The transport and ATPase cycle of Pgp is inhibited by phosphate-mimicking anions, such as orthovanadate (Vi), which can block the protein by stably replacing the cleaved gamma phosphate. The complex consisting of Pgp, ADP and vanadate (Pgp-ADP-Vi) is formed only under conditions that allow hydrolysis of at least one ATP molecule and is generally accepted to closely mimic the conformation of a transition state28. Of note, consistent with the formation of the Pgp-ADP-Vi complex, addition of vanadate increased the apparent affinity under hydrolysis conditions (37鈥壜癈, presence of ATP/Mg2+, Fig. 1a, Supplementary Table S1). However, when ATP hydrolysis was prevented, the apparent affinity was not influenced at all by Vi (Fig. 1b鈥揹), confirming that ATP hydrolysis and the subsequent release of the 纬-phosphate is a prerequisite for the formation of the Pgp-ADP-Vi complex29.Unilateral mutation of the Walker A lysine residues allows ATP binding and substrate-stimulated ATP hydrolysisThe Walker A motif (GXXGXGKS/T), also known as the P-loop (phosphate-binding loop), forms extensive bonds with the terminal phosphates of the bound nucleotide9. Mutation of the highly conserved lysine residue to methionine was shown to abolish ATPase activity of several ABC transporters30,31,32. To study the effect of mutations of the Walker A lysines in NBD1 (K433) and NBD2 (K1076) of Pgp on nucleotide-induced TMD conformational flexibility, we engineered mammalian cell lines (NIH 3T3 and MDCKII) stably expressing wild-type (WT), K433M, K1076M, or K433M/K1076M P-glycoprotein using a Sleeping Beauty transposon-based gene delivery system. Consistent with previous studies that showed that single Walker A mutations allow nucleotide binding32, UIC2-reactivity of the K433M and K1076M variants decreased in the presence of AMP-PNP and ATP (Fig. 2a鈥揷). Earlier we have shown the fluoroaluminate-dependent labeling of K433M and K1076M Pgp with [伪-32P]-8-azido-ATP33. Here we find that addition of vanadate results in a 5鈥?0-fold increase in the apparent affinity of the single mutants to ATP (Fig. 2b,c). Because complex formation with vanadate requires prior hydrolysis of ATP, these effects of vanadate indicate that Pgp is capable of ATP hydrolysis despite the mutation of a single Walker A lysine residue. In contrast, simultaneous mutation of both lysine residues resulted in a stable UIC2-binding conformation, which could not be reverted by nucleotides (Fig. 2d).Figure 2UIC2 mAb binding to single or double Walker A mutant Pgp variants in permeabilized cells in the presence of AMP-PNP/Mg2+ (a) or ATP/Mg2+ (b鈥揹) in the absence or presence of Vi. Permeabilized cells were treated with nucleotides at different concentrations for 20鈥塵in and then labeled with UIC2-A647 for another 30鈥塵in without washing out the nucleotides. In case of Vi treatment un-trapped nucleotides were washed out in the presence of Vi. UIC2-A647 fluorescence intensities were plotted as a function of nucleotide concentration and fitted with a four parameter Hill function to determine the apparent affinities of nucleotide binding (KA) and Hill slope values (see Materials and Methods).Full size imageThe turnover rate of Pgp鈥檚 catalytic cycle is accelerated in the presence of transported substrates (substrate-stimulated ATPase activity)27. Kinetic analysis of the formation of the BeFx- or Vi-trapped complexes showed that progressive accumulation in the low-UIC2-affinity trapped transition state occurs at comparable rates in WT or single mutant Pgps. Accumulation in the trapped complex was accelerated by Pgp substrates (e.g. verapamil, vinblastine, rhodamine 123) or competitive inhibitors (e.g. cyclosporine A) both in WT and single Walker A mutant Pgp variants (results are shown for vinblastine, see Fig. 3a,c,d), while the apparent affinity for ATP did not change even at high substrate concentrations (up to 200鈥壩糓 vinblastine, see Fig. 3b). The effect of transported substrates on the rate of formation of the trapped transition state complex was identical in the WT and the single mutant Pgp variants (Supplementary Table S3). These data suggest that despite the mutation of a single critical Walker A lysine residue, the catalytic cycle can progress to the step arrested by phosphate mimicking anions. The relatively long t1/2 values of BeFx (~120 s, see Fig. 3 and Supplementary Table S3) and Vi trapping (t1/2鈥?鈥?35.2鈥壜扁€?2.2 s; n鈥?鈥?) compared to the total cycle time which is on the order of 100鈥塵s suggest that formation of a stable post-hydrolysis complex by phosphate mimicking anions is an extremely low-probability event, likely reflecting the very short time window of the vanadate- or BeFx-鈥渟ensitive鈥?state in each cycle, that is between dissociation of the cleaved phosphate and disassembly of the NBD dimer. Thus, similarly to WT Pgp, single Walker A mutant Pgp variants can not only hydrolyze ATP, but pass on average ~1200鈥?500 cycles before trapping by BeFx or vanadate occurs. Consistently with this turnover, single mutants retain a weak, but significant verapamil stimulated ATPase activity that can be detected in membrane samples prepared from NIH 3T3 cells expressing the transporter at high levels (Supplementary Fig. S2).Figure 3Kinetics of the formation of the BeFx-trapped species in case of wild-type (a) and single mutant (c,d) Pgp expressing cells. Permeabilized cells were incubated with 100鈥壩糓 (for wild-type) or 500鈥壩糓 (for K433M and K1076M) ATP/Mg2+ and BeFx (200鈥壩糓 BeSO4 and 1鈥塵M NaF) in the presence or absence of 50鈥壩糓 vinblastine at 37鈥壜癈 and samples were taken at different time points. Panel (b) shows the effect of 200鈥壩糓 vinblastine on the apparent nucleotide affinity of wild-type Pgp. Permeabilized cells were treated with BeFx and different concentrations of ATP/Mg2+ in the presence or absence of 200鈥壩糓 vinblastine for 20鈥塵in at 37鈥壜癈. UIC2-A647 labeling was carried out following the washout of ATP/Mg2 in the maintained presence of BeFx.Full size imageThe rate of dissociation of the Vi-trapped complex is decreased by single Walker A mutationsAlthough the Vi-trapped species represent a dead-end of the catalytic reaction cycle, we wondered if the eventual release of the trapped nucleotide could be detected by an increase of UIC2 reactivity. Following the removal of uncomplexed Vi, complete time-dependent recovery of the UIC2-reactive state corresponding to the release of nucleotides was indeed observed (Fig. 4b). The half-life of Pgp-ADP-Vi complexes was 75.1鈥壜扁€?.9鈥塵in, in good agreement with literature data34,35. Release of the tightly bound nucleotide was temperature dependent (Fig. 4a,b), but was not influenced by the presence of ATP and transported substrates such as vinblastine or verapamil (not shown). UIC2 binding to the mutant Pgp variants was not restored in the time frame of the experiment (up to 4鈥塰ours), suggesting that mutation of a single Walker A residue increases the life time of the vanadate-trapped complex.Figure 4Recovery of UIC2 labeling of wild-type and single Walker A mutant Pgp.(a) Staphylococcus aureus alpha toxin-permeabilized NIH 3T3 cells expressing wild-type or single Walker A mutant Pgp were incubated with ATP and 500 渭M vanadate (Vi) to induce the formation of trapped Pgp-species. UIC2 mAb labeling was carried out after a 120-minute incubation at the indicated temperatures in Vi-free solution containing 50 渭M NEM in the absence of ATP. (b) Time-dependent recovery of the UIC2-binding conformation of wild-type Pgp. Aliquots washed for various time intervals in Vi- and ATP-free solution containing 200 渭M NEM were subsequently labeled with UIC2 mAb; washing and labeling were carried out either at 4鈥壜癈 or at 37鈥壜癈.Full size imageSingle Walker A mutants are transport-competentBecause single Walker A mutants were found to exhibit substrate-stimulated ATP hydrolytic activity (Fig. 3 and Supplementary Fig. S2), we next set out to determine whether they are also capable of promoting substrate transport. We compared the accumulation of calcein in both NIH 3T3 and MDCK cells expressing WT or mutant Pgp. Despite comparable expression levels, the Pgp-variant missing both Walker A lysine moieties (MM) was unable to hinder accumulation of calcein in the cells, whereas cells transfected with the single mutants showed a cyclosporine A-sensitive reduction in calcein fluorescence (Fig. 5a. and Supplementary Fig. S3). Reduction of intracellular calcein levels is a result of uphill transport activity which, by thermodynamic arguments, must be driven by ATP hydrolysis. To provide a quantitative measure of this residual transport activity, we determined the first order rate constants (k) of rhodamine123 (R123) efflux36 from cells expressing wild-type or mutant Pgp at comparable levels (Fig. 5b,c). In agreement with the data obtained with the calcein assay, single mutants showed significant R123 efflux activity, while the double mutant was inactive (Fig. 5d,e). In line with the relatively weak transport activity, single mutants conferred resistance against the cytotoxic effect of vinblastine (Supplementary Fig. S4). Collectively, these results suggest that single Walker A mutants retain a residual transport activity of ~15% of WT, whereas the double mutant is not transport competent.Figure 5Functional expression of wild-type and Walker A mutant human Pgp variants in MDCK cells.(a) Cells were incubated with 0.1鈥壩糓 calcein-AM with or without the Pgp-inhibitor cyclosporin A (CsA, 10鈥壩糓) for 30鈥塵inutes. Bars show the relative increase of calcein accumulation caused by CsA (means of 3 independent experiments鈥壜扁€塖D). (b) Mean fluorescence of Pgp-expressing cells stained with the anti-Pgp 15D3-A647 monoclonal antibody, showing comparable expression of Pgp variants in MDCK cells. *P鈥?lt;鈥?.05, **P鈥?lt;鈥?.01, ***P鈥?lt;鈥?.001. In panel (a) samples were compared to the Pgp non expressing MDCK cells, while in panel (b) the samples were compared to the wild-type Pgp (WT) expressing cells. (c) Western blot analysis of Pgp expression using a monoclonal anti-Pgp mAb (G-1, Santa Cruz Biotechnology Inc.). (d) Time dependent efflux of R123 from preloaded cells. Preloaded cells were incubated at 37鈥壜癈; during the first 5鈥塵in the fluorescence intensity of the cells was measured continuously and the values were averaged for 10 s intervals. At later time points fluorescence intensity distribution histograms were measured and their median fluorescence intensities were determined. (e) First order rate constants of the exponential curves fitted to the R123 efflux data (mean鈥壜扁€塖D of three independent experiments). Significant differences compared to the non-transfected MDCK cells are shown by **P鈥?lt;鈥?.01.Full size imageMutation of the critical Walker A lysines results in increased drug binding affinityConsistently with the Pgp-mediated efflux of vinblastine-bodipy (VBL-BPY), cells expressing WT Pgp show significantly lower levels of intracellular fluorescence as compared to Pgp鈭?/sup> cells when incubated with this fluorescent drug (Fig. 6, b vs. a). ATP-depleted cells overexpressing wild-type Pgp sequester VBL-BPY in the plasma membrane (Fig. 6c). Strikingly, plasma membrane sequestration of VBL-BPY was also observed in cells transfected with either single or double Walker A mutant Pgp variants (for double Walker A mutant see Fig. 6d). Addition of cyclosporine A (CsA), a competitive inhibitor of Pgp, prevented plasma membrane accumulation of VBL-BPY (Fig. 6e), suggesting that the enrichment of VBL-BPY in the plasma membrane is in each case due to its high-affinity binding to Pgp.Figure 6Accumulation of Bodipy FL Vinblastine (VBL-BPY) in the apical plasma membrane of energy deprived wild-type (WT) Pgp expressing and double Walker A mutant (K431M/K1076M) Pgp expressing MDCK cells reveals the stabilization of a high-affinity substrate binding Pgp conformation.VBL-BPY (green) and anti-Pgp 15D3-Alexa647 mAb (15D3-A647; red) staining of confluent non-transfected MDCK cells (a) wild-type Pgp-expressing (b,c) and Walker A mutant (K431M/K1076M) Pgp expressing MDCK cells (d,e). ATP depletion was induced by 30鈥塵in pre-treatment with 10鈥塵M Na-azide and 8鈥塵M 2-deoxyglucose treatment (c). A non-fluorescent competitive Pgp inhibitor CsA (10鈥壩糓) was added 10鈥塵in before VBL-BPY and antibody staining (e). Inserts show the apical membrane surface of one cell. Bar: 10鈥壩糾.Full size imageTo quantify the fraction of substrate-bound Pgp molecules in the membrane, Pearson鈥檚 cross-correlation coefficients were calculated between the VBL-BPY and 15D3 anti-Pgp antibody signals in single optical slices of apical membrane surfaces. Since the antibody staining was unchanged in the course of the treatments, the cross-correlation coefficients depend mostly on VBL-BPY binding to Pgp. In cells expressing double Walker A mutant Pgp (Fig. 7d) large cross-correlation coefficients of close to unity (~0.8) indicate that the majority of Pgp molecules are in high substrate affinity conformation. Single Walker A mutants also exhibited somewhat increased drug binding compared to the WT (Fig. 7b,c), but this binding was fully suppressed by transition state analogs, e.g. vanadate (Fig. 7b,c). Furthermore, ATP depletion by Na-azide or prevention of ATP binding by NEM treatment29 shifted both single mutant and WT Pgp variants into the substrate binding conformation, as reflected by increased co-localization of VBL-BPY and Pgp molecules in the plasma membrane (Fig. 7a鈥揷). Remarkably, the high substrate affinity of double mutant Pgp was not affected by any of the above treatments (Fig. 7d).Figure 7Pearson鈥檚 cross-correlation coefficients between the intensity distributions of cell-surface BODIPY FL Vinblastine (VBL-BPY) and 15D3-Alexa-647 labeling calculated for wild-type (a) single K433M (b); K1076M (c) and double Walker A mutant Pgp variants (d) expressed in MDCK cells. Cells were pre-treated for 30鈥塵in at 37鈥壜癈 with 10鈥壩糓 cyclosporine A (CsA), 2鈥塵M vanadate (Vi), 50 渭M N-ethyl-maleimide (NEM), or 10鈥塵M Na-azide and 8鈥塵M 2-deoxy D-glucose (ATP depletion) and then further incubated with VBL-BPY and 15D3-A647 anti-Pgp antibody for another 30鈥塵in at 37鈥壜癈. Significant differences compared to the wild-type鈥檚 untreated control are shown by ***P鈥?lt;鈥?.001, **P鈥?lt;鈥?.01, or *P鈥?lt;鈥?.05 and those compared to the untreated control of the respective cell line by: 鈥犫€犫€?/sup>P鈥?lt;鈥?.001, 鈥犫€?/sup>P鈥?lt;鈥?.01, or 鈥?/sup>P鈥?lt;鈥?.05. The untreated controls of the single and double mutants were also significantly different from each other (P鈥?lt;鈥?.01).Full size imageAMP-PNP binding is sufficient to switch Pgp from high to low drug affinity conformationIn permeabilized cells ATP depletion synchronized WT Pgp molecules in a high drug binding affinity conformation, as indicated by VBL-BPY sequestration in the plasma membrane (Fig. 8a, brown bar). Suppression of membrane fluorescence by CsA confirmed that drug accumulation in the membrane was indeed due to tight binding of VBL-BPY to Pgp (Fig. 8a, orange bar). This tight binding of VBL-BPY to Pgp was reversed in the presence of ATP and vanadate (Fig. 8a, ocher bar), consistent with the low drug binding affinity of the transition state complex37. Strikingly, incubation of permeabilized cells in 5 mM AMP-PNP also resulted in a significant decrease in the co-localization of VBL-BPY and Pgp, indicating that ATP hydrolysis is not required for the affinity switch responsible for the release of the transported substrate (Fig. 8a, last bar). AMP-PNP treatment resulted in a similar, although smaller, reduction of VBL-BPY binding to the single mutants (Fig. 8b). In contrast, simultaneous mutation of both Walker A lysine residues resulted in a permanent high drug binding affinity conformation, which could not be reversed by the addition of nucleotides (Fig. 8b).Figure 8BODIPY FL Vinblastine (VBL-BPY) binding to wild-type Pgp in permeabilized NIH 3T3 cells (a) and to Walker A mutant Pgps in permeabilized MDCK cells (b). Cells permeabilized by Staphylococcus alpha toxin (a) or Streptolysin O (b) were treated with different concentrations of CsA, ATP/Mg2++ vanadate (Vi) or AMP-PNP/Mg2+ followed by co-staining with VBL-BPY and 15D3-A647. The ratio of the VBL-BPY and 15D3-A647 fluorescence intensity was determined in pixels representing the plasma membrane, selected on the basis of 15D3-A647 fluorescence intensity exceeding the threshold of 300. Nucleotide-treated samples were compared to permeabilized ATP-depleted cells (0鈥塵M nucleotide); the values are means鈥壜扁€塖D of three independent experiments (***P鈥?lt;鈥?.001).Full size imageDiscussionBased on crystal structures and a wealth of biochemical and biophysical data it is generally accepted that ABC transporters adopt at least two discrete conformations along their transport cycle. The switch between the NBD-dissociated, inward-facing and the NBD-associated, outward-facing states involves a series of conformational changes which ultimately result in the reduction of substrate binding affinity required for uphill substrate transport. The exact molecular mechanisms that link nucleotide binding to the association and dissociation of NBDs and to the conformational changes of the TMDs are not fully understood. Our aim was to capitalize on the unique property of UIC2, a conformation-sensitive monoclonal antibody which recognizes a complex extracellular Pgp epitope, to follow the conformational changes which occur in the TMDs. UIC2 distinguishes two conformations of Pgp, which can be studied by modulating intracellular ATP-levels in Staphylococcus aureus alpha-toxin-permeabilized cells overexpressing Pgp25: the UIC2-dim and UIC2-reactive conformations correspond to the outward- and inward facing conformations observed in ATP-bound and nucleotide-free crystal structures, respectively. In addition, we monitored the nucleotide-dependent switch in drug binding affinity using a fluorescent Pgp substrate analog. Together, these assays allowed us to elucidate the link between ATP binding, hydrolysis and the conformational rearrangements responsible for switching the affinity of the substrate binding sites during substrate transport.The timing of the conformational change which results in the decrease of drug binding affinity has been a matter of much debate. The 鈥淎TP switch model鈥?predicts that ATP binding induces dimerization of the NBDs, which then triggers conformational changes resulting in the decrease of drug binding affinity; whereas the dissociation of the NBD resets the transporter for the next cycle38,39,40,41,42. In accordance with the above model the AMP-PNP-bound state of Pgp was reported to show reduced binding of vinblastine43,44. In some studies transition from the high to the low substrate affinity state was found to be triggered by nucleotide occlusion (i.e. tight binding) within the formed NBD dimer42,45,46. In contrast, other studies found that the conformational switch coupled to the change in drug binding affinity is driven by ATP hydrolysis47,48,49 or by the relaxation of a high-energy catalytic site conformation generated by the hydrolysis step28. These conflicting data may be due to the inherent limitations of the different experimental approaches such as the low labeling stoichiometry and the variable labeling efficiency of photoaffinity reagents, as well as the difficulty of quantifying binding affinity of highly lipophilic compounds to transmembrane transporters50,51.In view of the intimate association of Pgp with the lipid bilayer in which it is embedded and from which it harvests its substrates, it is worth noting that the confocal microscopic fluorescence co-localization assay and the UIC2 assay shown here characterize the transporter in its natural plasma membrane environment. Although the UIC2 assay is an indirect method for measuring the apparent affinities of nucleotides to Pgp, our experimental system is clearly capable of discriminating between distinct steps of the ATPase cycle such as nucleotide binding or ATP hydrolysis. Our results demonstrate that AMP-PNP binding is sufficient to induce the conformational switch corresponding to the transition from the inward to the outward facing conformation (Fig. 1c). Using the same experimental setup, we also show that binding of AMP-PNP switches Pgp into the low drug binding affinity state (Fig. 8a). The simultaneous drop in the UIC2- and drug-binding affinities suggests that the high-to-low switch in drug binding affinity might coincide with the transition from the inward- to the outward-facing conformation, but in any case precedes ATP hydrolysis (Figs 1,2 and 8.).Residues of the Walker motifs in each NBD, together with the signature sequence of the contralateral NBD, directly participate in nucleotide-dependent dimerization of the two NBDs and ATP hydrolysis. How ATP hydrolysis is coordinated between the two NBDs and whether Pgp hydrolyses one or two (or more) ATP molecules per each transported substrate are not known. In diverse ABC transporters, mutations of the conserved Walker A lysine reduce ATPase activity to very low levels30,31,32,52,53,54. The two nucleotide binding domains of Pgp were shown to be functionally equivalent and the integrity of both catalytic centers is generally believed to be needed for transport, because inactivation of a single NBD results in inhibition of ATPase and transport activities29,34,55.We confirm that mutation of both Walker A lysine residues inactivates Pgp (Fig. 5 and Supplementary Fig. S3): as indicated by the lack of ATP-triggered conformational changes, the transporter is essentially frozen in the UIC2-reactive inward-open state (Fig. 2) characterized with high drug binding affinity (Figs 6, 7, 8). Intriguingly, however, our data show that nucleotide binding to single Walker A mutants triggers the same inward-to-outward conformational switch (Fig. 2a) and the concomitant drop in drug binding affinity (Figs 7 and 8), as observed for the WT protein. Furthermore, we demonstrate that vanadate exerts the same effect on the UIC2 binding curves of the WT and the unilateral Walker A mutant Pgp variants. Since vanadate-dependent trapping is an extremely low-probability event, formation of the stable Vi-trapped complex (Fig. 3), as well as the significant left shift of the UIC2 binding curves in the presence of Vi (Fig. 2b,c), can only be explained if single Walker A mutants are catalytically competent. Admittedly, this result is opposed to prevailing views insisting on the requirement of two intact nucleotide binding domains for ATP hydrolysis by Pgp28. While our experimental system has several limitations (binding and hydrolysis of ATP is measured indirectly, by monitoring the binding of a conformation sensitive antibody to a complex extracellular epitope of Pgp in semipermeabilized cells), we note that the majority of the studies reporting the inactivating effect of single Walker A mutations have been performed using heterologous expression systems such as Sf932,56, Saccharomyces cerevisiae57 or purified and reconstituted proteins52,57,58. It is known that the plasma membrane composition influences the catalytic activity of ABC transporters and that membrane cholesterol amounts influence Pgp activity59,60. Since membrane cholesterol levels are significantly lower in lower eukaryotes60,61, it may be that the low ATPase activity of the single Walker mutants was missed due to the different plasma membrane composition of the heterologous expression systems, or artefacts related to the solubilization, purification and reconstitution of the proteins62. We show that the single Walker A mutants show drug-stimulated nucleotide hydrolysis (Fig. 3 and Supplementary Fig. S2), decrease the intracellular accumulation of calcein (Fig. 5a), promote R123 efflux (Fig. 5d鈥揺) and offer moderate protection against cytotoxic Pgp substrates (Supplementary Fig. S4). Taken together, our data indicate that, in contrast to prevailing views, single-site Walker A mutant Pgp molecules retain a weak, but significant uphill transport activity, a phenomenon which cannot be explained other than through coupling to repeated cycles of ATP hydrolysis.Formation of the vanadate-trapped species occurs with comparable rates in WT and single mutant Pgp (Fig. 3), suggesting that the fraction of time the working transporter spends in the Vi-sensitive posthydrolytic state is similar for WT and single mutant Pgp. Interestingly however, the rate of disassembly of the Vi-trapped complex is greatly reduced in the single mutants (Fig. 4a). Assuming that the trapped complex harbors an ATP in the non-committed site, one might speculate that disassembly of the complex happens only upon eventual hydrolysis of that ATP. This process might happen at a rate of ~1 per hour in a non-committed wild-type catalytic center (cf., Fig. 4b), but never at all in a center disabled by a Walker A mutation (Fig. 4a). Taken together our experiments carried out with single Walker A mutants suggest that while one hydrolysis event is sufficient to reset the high-substrate affinity inward facing conformation in the absence of Vi, dissociation of the Vi-trapped complex also requires the hydrolytic activity of the contralateral site, supporting the possibility that the Vi-trapped complex may not mimic a true catalytic transition state in case of Pgp. However, much further work will be required to fully understand these details.Importantly, the fact that single Walker A mutations allow residual transport is incompatible with a model in which two ATP molecules must be hydrolyzed in each cycle. Our results are also difficult to reconcile with models suggesting that the two NBDs hydrolyze ATP in a strictly alternating order28,47. If the NBDs were indeed recruited in a strictly alternating fashion, every second ATP would have to be processed by the mutant catalytic center, causing the cycle to stall. Instead, our results indicate that the wild-type catalytic site can hydrolyze ATP in repeated cycles without hydrolysis at the other NBD. The simplest interpretation of our data is that in WT Pgp one of the two functionally equivalent sites becomes committed to hydrolysis in each cycle on a random basis, whereas in the single mutants commitment of the only functional site initiates every cycle.Finally, a simplified kinetic model that distinguishes inward- and outward-facing states (Fig. 9; see full mathematical treatment in Supplementary Information) provides a semi-quantitative framework for interpretation of the data presented here and offers some additional mechanistic insight. The model includes ATP-free (X0) and ATP-bound (X1) inward-facing states (associated with an open NBD dimer), and, for simplicity, depicts ATP binding as a single step assumed to be at rapid equilibrium. From the ATP-bound inward-facing conformation the transporter flips (with rate k1) to the ATP-bound outward-facing X2 state (associated with a closed NBD dimer). The model is very general, in that it does not specify (dotted arrow) the number and nature of intermediate states between X2 and the final post-hydrolytic outward-facing state Xn, which eventually flips back to inward-facing state X0 upon NBD-dimer dissociation and release of hydrolysis products. Because UIC2 reactivity is believed to be uniformly low in all outward-facing states, the model allows UIC2 binding only to inward-facing conformations X0 and X1, yielding UIC2-bound states X0* and X1*, respectively. For simplicity, binding of UIC2 and ATP are assumed independent 鈥?consistent with the fact that UIC2 recognizes an extracellular epitope22 formed by the TMDs, the conformation of which is unlikely to sense mere binding of ATP to intracellular NBDs. The parameters of the model are as follows:Figure 9Simplified model of the catalytic cycle of Pgp that distinguishes inward- and outward-facing states.Full size imageKd;ATP is the dissociation constant of ATP from inward-facing Pgp,Kd;UIC2 is the dissociation constant of the UIC2 antibody from inward-facing Pgp,T1 is the average life time of the inward-facing conformation,i.e., of compound state {; ; ; },T1;min (=1/k1) is T1 in the presence of zero [UIC2] and saturating [ATP], andT2 is the average life time of the outward-facing conformation, i.e., of compound state {;鈥?}.v, is the turnover rate for ATP hydrolysis (or substrate transport), which is the inverse of the total cycle time T (=T1鈥?鈥?i>T2).Despite its simplicity, the model is congruent with a broad range of existing data. (i) Consistent with the literature48,63, the scheme (Fig. 9) predicts that ATP-hydrolysis and ATP-dependent substrate transport by Pgp follow Michaelis-Menten type kinetics (see Supplementary Information Eqs 3). (ii) It further predicts that UIC2 should act as a mixed type inhibitor of Pgp-mediated ATP-hydrolysis and substrate transport, by lowering the apparent Vmax and by increasing the apparent KM for ATP (see Supplementary Information Eqs 1, 2 and 5鈥? for details). Indeed, UIC2 inhibits the efflux of Pgp substrates from multidrug resistant cells and significantly increases the cytotoxicity of Pgp-transported drugs64,65. (iii) Consistent with our finding that ATP shifts Pgp into a low UIC2-affinity conformation in a concentration dependent manner (Fig. 1a), the scheme (Fig. 9) predicts that increasing [ATP] should diminish UIC2-labeling (by a fixed concentration of UIC2) following a Michaelis-Menten-type dose-response relationship and the apparent affinity of ATP for competing UIC2 labeling (KA) should be identical to the KM of ATP for ATP-hydrolysis and substrate transport observable at the same fixed [UIC2] (see Supplementary Information Eq. 7). (iv) Finally, the model predicts that the apparent affinity for UIC2 binding (KU) should be sensitive to ATP concentration (see Supplementary Information Eqs 9鈥?1), which was verified in UIC2 titration experiments (Supplementary Fig. S5). These features qualified the model to be suitable for semi-quantitative interpretation of our results and allowed us to draw some additional mechanistic conclusions.First, our experiments provide two independent estimates of the fraction of time wild-type Pgp spends in the outward- vs. inward facing conformations under normal, hydrolytic conditions in saturating ATP (i.e., the ratio T2/T1;min). The first approach builds on the maximal shift in apparent affinity for UIC2 binding (KU) caused by very high concentrations of ATP. In the experiments documented in Supplementary Fig. S5 we determined KU at various different [ATP]. In the absence of ATP, KU reflects the antibody鈥檚 dissociation constant (Kd;UIC2), which was found to be ~2鈥壩糶/ml (~10鈥塶M). In contrast, at very high [ATP], KU approaches ~20鈥壩糶/ml 鈮?10Kd;UIC2. This maximal ~10-fold shift in KU by high [ATP] indicates that in saturating [ATP] Pgp spends 90% of the total cycle time in the outward-facing (UIC2-dim) conformation (, see Supplementary Information Eq. 11). An independent estimate of T2/T1;min is obtained from the fraction of UIC2 label which resists even at saturating [ATP]. Applying a fixed [UIC2] of 10鈥壩糶/ml, equivalent to 鈮?Kd;UIC2, we found that approximately one third of the UIC2 label persists at very high [ATP] (Fig. 1a), from which Eq. 8 (see Supplementary Information) predicts a ratio T2/T1;min of ~12. Thus, both approaches suggest that, in saturating ATP, WT Pgp spends the majority ( 90%) of the total cycle time in the outward-facing conformation (compound state {;鈥?} in the scheme (Fig. 9)). With other words, flipping from the ATP-bound inward-facing to the ATP-bound outward-facing conformation (step X1鈥夆啋鈥?i>X2 in Fig. 9) is not the rate-limiting step of the overall cycle. A direct implication of this finding is that in Pgp substrate-mediated stimulation of ATPase turnover rate must reflect acceleration of some other step(s): mere speeding of step X1鈥夆啋鈥?i>X2 could increase turnover rate by no more than ~10%. It will be interesting to establish how broadly this conclusion is applicable to other members of the ABC protein family. For instance, in the CFTR chloride ion channel (ABCC7) pore opening, believed to correspond to the transition from inward- to outward-facing, is no doubt the slowest step of the gating cycle and is most robustly affected by channel phosphorylation, the primary mechanism for regulating CFTR activity (reviewed in66).Second, the model allows comparison of the kinetics of the transport cycles of Walker A single mutants to that of WT. From the increased fraction of UIC2 label persisting in saturating ATP (~50%, Fig. 2b,c) we estimate a decreased ratio of(Supplementary Information Eq. 8), suggesting that in saturating [ATP], the fraction of time spent in the outward-facing conformation is somewhat decreased for single Walker A mutants relative to WT. This explains on one hand the higher apparent affinities for substrate binding observed for the single mutants (Fig. 7b,c vs. Fig. 7a; bars labeled 鈥渃ontrol鈥?, but in part also the more modest suppression of substrate affinities by 3鈥?鈥塵M AMP-PNP (Fig. 8b; note also that for the single mutants 3鈥?鈥塵M AMP-PNP is less than saturating (Fig. 2a and Supplementary Table S2)). In itself, the ~10 fold decrease in apparent affinity for ATP of the single mutants (Fig. 2b,c and Supplementary Table S2) would explain only a modest decrease in transport rate (to 80鈥?0% of WT) at physiological ATP concentrations. Thus, to account for a transport rate which is only ~10鈥?5% of WT (Fig. 5), the average overall cycle time T of the single mutants must be significantly prolonged even in saturating ATP. Given our above estimate of T1;min/T2 鈮?10 for WT-Pgp, a mere increase in T1;min (to yield ), would prolong the overall cycle time T only by ~6%: this falls far short of explaining the robust decline in transport rate (see Supplementary Information for more details). Two possible scenarios could account for this discrepancy. One possibility is that both T1;min and T2 are prolonged: in this view all cycles would go through ATP hydrolysis at the competent site, but both the rate of formation of the NBD dimer (k1) and the rate of ATP hydrolysis (step X2鈥夆啋鈥夆€?Xn, overall rate k2) would be slowed. Alternatively, besides allosterically slowing ATP hydrolysis at the competent site, the major disturbance caused by single mutations could be destabilization of the NBD dimer, i.e., an increase in the slow rate (k-1) of reverse step X2鈥夆啋鈥?i>X1. As a consequence, only ~1 out of 6 sojourns in the X2 state would be terminated by ATP-hydrolysis (鈥減roductive鈥?transport cycles), whereas in ~5 out of 6 cases the reverse pathway would be taken (鈥渇ruitless鈥?partial cycles)鈥搘ith little change in the cycle time T itself. A similar mechanism, i.e., ~20% coupling between ATP-hydrolysis and pore opening events, has been suggested for NBD1 Walker-A mutant K464A CFTR channels67.In conclusion, we have shown that the high-to-low substrate affinity switch in Pgp coincides with the flipping of the TMDs to the outward-facing conformation and that these events precede ATP hydrolysis. Double Walker A mutant Pgp is trapped in the high-affinity inward-facing conformation, but single mutants are capable of full transport cycles albeit with reduced efficiency. Release from the vanadate-trapped low-affinity outward-facing conformation requires two functional composite sites. These results support random recruitment of the two catalytic centers for ATP hydrolysis.Materials and MethodsChemicalsCell culture media, supplements and chemicals were from Sigma-Aldrich (Budapest, Hungary). Fluorescent dyes including calcein acetoxymethyl ester (calcein-AM), BODIPY FL vinblastine (vinblastine-bodipy; VBL-BPY) and Alexa 647 succinimidyl ester were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). The UIC2 and 15D3 anti-Pgp mAbs were prepared from hybridoma supernatants using affinity chromatography and were 97% pure by SDS/PAGE. Hybridoma cell lines were obtained from the American Type Culture Collections (Manassas, VA, USA). The UIC2 and 15D3 antibodies were labeled with Alexa 647 succinimidyl ester (A647) and separated from the unconjugated dye by gel filtration on a Sephadex G-50 column. The dye-to-protein labeling ratio was around 3 for each antibody preparation.Cell linesThe NIH 3T3 mouse fibroblast cell line was a kind gift from Michael Gottesman (National Institutes of Health, Bethesda, MD). The MDCK II Madin-Darby canine kidney cell line was obtained from Balazs Sarkadi (Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary). The cells were grown as monolayer cultures at 37鈥壜癈 in an incubator containing 5% CO2 and were maintained by regular passage in Dulbecco鈥檚 modified Eagle鈥檚 medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 2鈥塵M L-glutamine and 0.1鈥塵g/ml penicillin-streptomycin cocktail.Vector constructsSleeping Beauty transposon vectors containing the wild-type (WT), K433M, K1076M or the K433M/K1076M double mutant human MDR1 cDNA were constructed as follows. Site-directed mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) on pAcUW-LMDR1 vector carrying the wild-type human MDR1 cDNA. Mutations were generated according to the manufacturer鈥檚 instructions. The following oligonucleotide primers were used: 5鈥?AAACAGTGGCTGTG GGATGAGCACAACAGTCCAGCTGA-3鈥?and 5鈥?CAGCAGTGGCTGTGGGATGAGC ACAGTGGTCCAGCTCC-3鈥?for the K433M and K1076M mutations, respectively. The Sleeping Beauty (SB) transposon vector containing the cDNAs of EGFP and the puromycin resistance gene (PURO) in separate transcription cassettes, each driven by CAG promoter, was kindly provided by Dr. Tam谩s Istv谩n Orb谩n (Hungarian Academy of Sciences, Budapest, Hungary). Full-length wild-type MDR1 cDNA was amplified by PCR from pAcUW-LMDR1 vector with the following pair of primers: 5鈥?TAGAATACCGGTAGGTCGGAATGGATCTTGAA-3鈥?and 5鈥?AGTGATGGATCCAACATCTCATACAGTCAGAG-3鈥?containing AgeI and BamHI restriction sites, respectively. The digested PCR product was cloned into the SB-EGFP-PURO transposon vector between the AgeI and BclI restriction sites, replacing EGFP, resulting in SB-MDR1-PURO vector. The PstI-BstBI restriction fragment of the MDR1 cDNA was then replaced in SB-MDR1-PURO with the mutated PstI-BstBI fragments derived from pAcUW-LMDR1K433M and pAcUW-LMDR1K1076M, resulting in SB-MDR1K433M-PURO and SB-MDR1K1076M-PURO vectors. The SB construct containing the K433M/K1076M double mutant MDR1 cDNA was generated by replacing the 3242bp long HindIII-HindIII fragment in SB-MDR1K1076M-PURO with that of SB-MDR1K433M-PURO, resulting in SB-MDR1K433M/K1076M-PURO. Full-length MDR1 cDNAs were sequenced and mutations were confirmed in all SB constructs.Establishment of transgenic cell linesCell lines stably expressing WT, K433M, K1076M and K433M/K1076M double mutant MDR1/P-glycoprotein were established by the Sleeping Beauty (SB) transposon-based gene delivery system, using the 100 fold hyperactive SB transposase68. NIH 3T3 mouse fibroblast and MDCK II canine kidney cells were co-transfected with the SB transposase and SB transposon vector constructs by Lipofectamine2000 reagent (Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer鈥檚 instructions. Briefly, 3鈥壝椻€?05 cells were seeded in 6-well-plates, 24鈥塰ours later cells were transfected with 2鈥壩糶 vector DNA per well in a 10:1 ratio for the SB transposon and transposase constructs. 48鈥塰ours after transfection transgene positive cells were sorted by flow cytometry (FACS Aria High Speed Cell Sorter, Becton Dickinson) based on the cell surface expression of wild-type and mutant MDR1/P-glycoprotein. Protein expression was measured by antibody labeling using the human MDR1/P-glycoprotein specific monoclonal antibodies MRK16 (Abnova GmbH, Heidelberg, Germany) or 15D3. To obtain homogenously expressing cell populations, sorting procedure was repeated 2 weeks after transfection.Western blot analysisTotal cellular protein (2.5鈥壩糶/slot) was subjected to SDS-polyacrylamide gel electrophoresis on 8% polyacrylamide gel and electro-blotted to 0.45鈥壩糾 pore size nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). Pgp expression was detected by a monoclonal anti-Pgp mAb (G-1, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and a goat anti-mouse HRP-conjugated IgG secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), both applied at 1:5,000 dilution.Permeabilization of cells with Staphylococcus aureus 伪-toxin or streptolysin-OStaphylococcus aureus 伪-toxin and streptolysin-O (Sigma-Aldrich, Budapest, Hungary) binds to the plasma membrane of cells and forms ring-structured toxin hexamers that are permeable for small water soluble molecules including nucleotides69. Cell suspensions (1鈥壝椻€?07 cells/ml) were treated with 4鈥壩糶/ml 伪-toxin in phosphate-buffered saline (PBS) in the presence of 1% bovine serum albumin (BSA) at 37鈥壜癈 for 30鈥塵in, allowing permeabilization of approximately 50% of the cells (judged by propidium iodide (PI) positivity)25. The reaction was stopped with 40鈥塵l of 37鈥壜癈 PBS and the cells were centrifuged for 7鈥塵in at 635鈥塯 at room temperature. Unbound toxin was removed by washing the cells 3 times with PBS and the cell pellet was re-suspended in PBS.Cells grown attached to the bottom of Ibidi 渭-Slide IV 0.4 (for confocal microscopy) were permeabilized by 50鈥壩糶/ml Staphylococcus aureus 伪-toxin or 20鈥壩糶/ml (approx. 1300鈥塙/ml) streptolysin-O in the presence of 10鈥塵M DTT and Protease Inhibitor Cocktail at 37鈥壜癈 for 60鈥塵in in HEPES buffer (20鈥塵M HEPES, 123鈥塵M NaCl, 5鈥塵M KCl, 1.5鈥塵M MgCl2, 1鈥塵M CaCl2) containing 1% FCS (allowing permabilization of 60鈥?0% of cells).Determination of the apparent affinity of nucleotide bindingPermeabilized cells (1鈥壝椻€?06 ml鈭?) were pre-incubated for 20鈥塵in with nucleotides added in a broad concentration range and then further incubated for 30鈥塵in with 10鈥壩糶/ml A647-conjugated UIC2 monoclonal antibody (all at 37鈥壜癈). UIC2 mAb binding to Pgp is a reversible reaction in the presence of ATP/Mg2+ (Supplementary Fig. S6), thus, UIC2 was applied at a quasi-saturating concentration (5Kd;UIC2; Supplementary Fig. S5) and the duration of the incubation period was sufficient to reach equilibrium binding of UIC2 (Supplementary Fig. S6).When ATP was applied under hydrolysis conditions the concentration of the cells was decreased to 1.5鈥壝椻€?04 ml鈭? in order to reduce the consumption of ATP. Under these conditions the activity of Pgp and endogenous ATPases present in the permeabilized cells did not significantly reduce the concentration of ATP in the samples (less than 3% of the ATP was converted to ADP).To prevent ATP hydrolysis, ATP was added to permeabilized cells without Mg2+ in the presence of 5鈥塵M EDTA. Alternatively, ATP was replaced by the non-hydrolysable ATP analogue AMP-PNP/Mg2+, or the experiment was conducted on ice. In the latter case the labeling of cells with A647-conjugated UIC2 monoclonal antibody was carried out at 4鈥壜癈 for 45鈥塵in. Following antibody labeling, samples were washed 3 times with ice-cold PBS and centrifuged for 7鈥塵in at 635鈥塯 at 4鈥壜癈. The UIC2-A647 fluorescence intensity of the cells was measured by flow cytometry and plotted as a function of the nucleotide concentration. To determine the apparent affinity of Pgp to the nucleotides (KA) data points were fitted with the four-parameter Hill function, where Fmin and Fmax values are the minimum and maximum fluorescence intensities:Nucleotide trappingThe transition state of Pgp was stabilized by the addition of ATP/Mg2+ and vanadate (Vi) or BeFx at 37鈥壜癈. Permeabilized cells were incubated with 100鈥壩糓 (for WT) or 500鈥壩糓 (for K433M and K1076M) ATP/Mg2+ and 0.5鈥塵M Vi70 or BeFx (200鈥壩糓 BeSO4 and 1鈥塵M NaF) in the presence or absence of substrates in PBS at 37鈥壜癈. 500鈥壩糽 aliquots were taken at regular intervals and washed twice with 5鈥塵l ice-cold PBS containing Vi or BeFx at the same concentrations that were applied previously during incubation. Samples were re-suspended in ice-cold PBS and labeled with 10鈥壩糶/ml UIC2-A647 monoclonal antibody in the presence of the transition state analogue at 4鈥壜癈 for 45鈥塵in. The UIC2-A647 fluorescence intensity of the samples (F) was plotted as a function of time (t). The t1/2 values representing the half-life of the UIC2-reactive Pgp conformation were calculated from an exponential fit of the data points according to the following equation:where Fo i.e. the difference between the zero and infinite time point of the curve and c is the background fluorescence intensity of the cells.Recovery from the Vi-trapped post-hydrolysis statePermeabilized cells trapped in the presence of ATP/Mg2+ (100鈥壩糓 for wild-type and 500鈥壩糓 for single Walker A mutants) and 0.5鈥塵M Vi were washed 3 times with ice-cold PBS and incubated in the presence or absence of ATP and/or substrates (verapamil, vinblastine) at 37鈥壜癈. 500鈥壩糽 aliquots containing 2鈥壝椻€?05 cells were taken at regular intervals and washed twice with 5鈥塵l PBS. The aliquots were incubated in the presence of 200 渭M N-ethyl-maleimide ((NEM) to avoid de novo disulphide bond formation between Walker A cysteines71) and labeled with 10 渭g/ml UIC2-A647 at 37鈥壜癈 for 15鈥塵in or at 4鈥壜癈 for 45鈥塵in. At the end of incubations the samples were washed three times with ice-cold PBS and kept on ice until measurement in the flow cytometer.Calcein assayCalcein accumulation was measured as described70. Briefly, cells (0.25鈥壝椻€?06 ml鈭?) were pre-incubated with a Pgp modulator cyclosporine A (CsA, 10 渭M) for 10鈥塵in and then further incubated with 100鈥塶M calcein-AM at 37鈥壜癈 for 30鈥塵in. Finally, samples were washed with ice-cold PBS containing 1% FCS (FCS-PBS) and kept on ice until measurement. Dead cells stained with PI were excluded from the analysis. Transport activity was described by the ratio of median fluorescence intensities of the CsA-treated and untreated samples.Rhodamine 123 efflux assayCells (1鈥壝椻€?06 ml鈭?) were pre-loaded with 0.5 渭M rhodamine 123 (R123) for 30鈥塵in at 37鈥壜癈. Loading was terminated by chilling the tubes on ice; cells were washed three times with ice cold PBS (containing 8鈥塵M glucose and 1% FCS, (gl-FCS-PBS), pH鈥?鈥?.4). Efflux was initiated by re-suspending the cell pellet in gl-FCS-PBS pre-warmed to 37鈥壜癈. Efflux was monitored continuously over 5鈥塵inutes with a Becton Dickinson FACS Aria III flow cytometer at 37鈥壜癈 using a temperature controlled sample injection chamber. Cells were further incubated at 37鈥壜癈 and samples were taken at 10, 20, 30, 60 and 120鈥塵in time points and their median fluorescence intensity was measured using the same settings of the cytometer. Viable cells were selected by PI exclusion. Upon continuous monitoring of R123 efflux mean fluorescence intensities were computed for 10 second time intervals. Median fluorescence intensities were displayed as a function of time and the first order rate constants (k-values) were calculated from an exponential fit of the data points according to Eq. 3, where a is initial loading (i.e. the difference between the zero and infinite time point of the curve), t is the time in minutes,is mean fluorescence intensity at t time point and c is background fluorescence intensity of the cells.Vinblastine-bodipy stainingCells were plated in 8-well Ibidi 渭-Slide (Ibidi GmbH, Martinsried, Germany) two days before the experiments to obtain nearly confluent cultures. The samples were pre-incubated with the following agents: Na-azide (10鈥塵M) and 2-deoxy D-glucose (8鈥塵M), Vi (2鈥塵M) or NEM (50 渭M) for 30鈥塵in and then further incubated with 100鈥塶M vinblastine-bodipy (VBL-BPY) and 30 渭g/ml A647-conjugated 15D3 anti-Pgp antibody for another 30鈥塵in at 37鈥壜癈.Flow cytometryCalcein accumulation and R123 efflux measurements were carried out on a Becton Dickinson FACSAriaTM III Cell Sorter (Becton Dickinson, Mountain View, CA, USA). Calcein and R123 were excited by the 488鈥塶m line of a solid state laser and the emitted light was detected using a 502鈥塶m dichroic mirror and a 530/30鈥塶m band-pass filter. PI was excited by the 562鈥塶m line of a solid state laser and the emitted light was detected applying a 590鈥塶m dichroic mirror and a 595/50鈥塶m band-pass filter. In case of continuous monitoring of R123 efflux the sample injection chamber was kept at 37鈥壜癈, while in other cases it was at 4鈥壜癈. Cytofluorimetric data were analysed by using FCS Express 4 Research Edition (De Novo Software, Glendale, CA, USA).UIC2-A647 labeling of permeabilized cells was measured by using a Becton Dickenson FACS Array flow cytometer; the data were analysed with the BDIS CellQuest software. A 635鈥塶m laser was used for the excitation of the Alexa 647 dye and the fluorescence was detected in the red channel (661/16鈥塶m), while the 532鈥塶m laser was used for the excitation of PI (detected at 585/42鈥塶m). Cell debris was excluded from analysis on the basis of FSC and SSC signals. The median fluorescence indicating UIC2 reactivity was determined in PI positive cells.Confocal laser scanning microscopyCellular localization of Pgp and VBL-BPY staining was studied by a three-laser confocal laser scanning microscope (Olympus FluoView 1000 confocal microscope based on an inverted IX-81 stand with an UPlansAPo 60鈥壝椻€塏A 1.35 oil immersion objective, Hamburg, Germany). The 488-nm blue line of an argon-ion laser and the 543-nm green and the 633-nm red helium-neon laser lines were used for the excitation of VBL-BPY, PI and Alexa 647, respectively. Fluorescence intensities were detected in the spectral ranges of 500鈥?30鈥塶m, 555鈥?55鈥塶m and 655鈥?55鈥塶m, respectively. To assess the co-localization of VBL-BPY (green) and Alexa 647-conjugated 15D3 (red) fluorescence signals, single optical slices of apical membrane surfaces were recorded for double-labeled cells. The pattern and extent of co-localization in the sections were determined by using the cross-correlation scattergrams of the green and red pixels of the images. Co-localization indices were determined from 30鈥?0 cells (in three independent experiments). The Pearson鈥檚 co-localization index (CI) provides a reliable estimate on the extent of fluorescence co-localization: CI values close to zero indicate no or a very low degree, while CI鈥夆墺鈥?.5 reflects a high degree of co-localization, whereas the CI鈥?鈥? value would correspond to a full overlap between the two colours in each pixel of the image.In case of permeabilized cells VBL-BPY flurescence intensity was determined in pixels representing the apical plasma membrane selected on the basis of 15D3-Alexa-647 fluorescence intensity exceeding the threshold of 300 above background intensity.Statistical analysisData were analysed using SigmaStat (version 3.1, SPSS Inc., Chicago, IL, USA) and are presented as means鈥壜扁€塖D. Comparison of two groups was carried out by unpaired t-test, statistical significance in the case of three or more groups was assessed using analysis of variance (ANOVA), applying the Holm-Sidak multiple comparison test for post hoc pair-wise comparison of the data. 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GSzak谩cs and LCs were supported by a Momentum Grant of the Hungarian Academy of Sciences. Funding from ERC (StG-260572), NKTH-ANR 10-1-2011-0401 and the Austrian Science Fund SFB35 (GSzak谩cs) is also acknowledged.Author informationAuthor notesB谩rsony Orsolya and Szal贸ki G谩bor contributed equally to this work.Szak谩cs Gergely and Goda Katalin jointly supervised this work.AffiliationsDepartment of Biophysics and Cell Biology, University of Debrecen, P.O. Box 400, Debrecen, H-4002, HungaryOrsolya B谩rsony,聽G谩bor Szal贸ki,聽Szabolcs Tarapcs谩k,聽Zsuzsanna Gutay-T贸th,聽Zsolt Bacs贸,聽G谩bor Szab贸聽 聽Katalin GodaInstitute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, HungaryD贸ra T眉rk聽 聽Gergely Szak谩csInstitute of Horticulture, University of Debrecen, P.O. Box 36, Debrecen, H-4015, HungaryImre J. HolbPlant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, H-1525, HungaryImre J. HolbDepartment of Biochemistry and Molecular Biology, MTA-DE Momentum, Genome Architecture and Recombination Research Group, University of Debrecen, Debrecen, H-4032, HungaryL贸r谩nt Sz茅kv枚lgyiMTA-SE Ion Channel Research Group, Budapest, H-1094, HungaryL谩szl贸 Csan谩dyDepartment of Medicine I, Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Vienna, AustriaGergely Szak谩csAuthorsOrsolya B谩rsonyView author publicationsYou can also search for this author in PubMed聽Google ScholarG谩bor Szal贸kiView author publicationsYou can also search for this author in PubMed聽Google ScholarD贸ra T眉rkView author publicationsYou can also search for this author in PubMed聽Google ScholarSzabolcs Tarapcs谩kView author publicationsYou can also search for this author in PubMed聽Google ScholarZsuzsanna Gutay-T贸thView author publicationsYou can also search for this author in PubMed聽Google ScholarZsolt Bacs贸View author publicationsYou can also search for this author in PubMed聽Google ScholarImre J. HolbView author publicationsYou can also search for this author in PubMed聽Google ScholarL贸r谩nt Sz茅kv枚lgyiView author publicationsYou can also search for this author in PubMed聽Google ScholarG谩bor Szab贸View author publicationsYou can also search for this author in PubMed聽Google ScholarL谩szl贸 Csan谩dyView author publicationsYou can also search for this author in PubMed聽Google ScholarGergely Szak谩csView author publicationsYou can also search for this author in PubMed聽Google ScholarKatalin GodaView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsO.B. designed and performed UIC2 reactivity measurements. G.Szal贸ki designed and performed drug efflux assays and microscopic experiments. D.T. generated the vector constructs and transgenic cell lines. S.T. carried out cytotoxicity assays and ATPase activity measurements. Z.G.-T. and Z.B. carried out UIC2 dissociation assays. I.J.H. and G.Szal贸ki carried out the statistical analysis of the data. L.S. took part in designing the experiments and carried out PCR experiments. L.C. generated and interpreted the mathematical model and took part in the preparation of the manuscript. K.G. and G.Szak谩cs prepared the manuscript, conceived most of the experimental ideas and directed the work. G.Szab贸 co-directed the initial phase of the research project together with K.G.Ethics declarations Competing interests The authors declare no competing financial interests. Electronic supplementary material Supplementary InformationRights and permissions This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article鈥檚 Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and PermissionsAbout this articleCite this articleB谩rsony, O., Szal贸ki, G., T眉rk, D. et al. A single active catalytic site is sufficient to promote transport in P-glycoprotein. Sci Rep 6, 24810 (2016). https://doi.org/10.1038/srep24810Download citationReceived: 05 October 2015Accepted: 05 April 2016Published: 27 April 2016DOI: https://doi.org/10.1038/srep24810 P茅rez-De Marcos Juan-Carlos, P茅rez-Pineda Perla-Lidia, M茅ndez-Morales Stephanie-Talia, Arellano-Mendoza M贸nica-Griselda Torres-Esp铆ndola Luz-Mar铆a Molecular Biology Reports (2021) Iva Guberovi膰, Marko Marjanovi膰, Marija Mio膷, Katja Ester, Irena Martin-Kleiner, Tatjana 艩umanovac Ramljak, Kata Mlinari膰-Majerski Marijeta Kralj Scientific Reports (2018) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. 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