Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

THE TWO ATP HYDROLYSIS EVENTS IN A SINGLE CATALYTIC CYCLE ARE KINETICALLY SIMILAR BUT AFFECT DIFFERENT FUNCTIONAL OUTCOMES*

Zuben E. Sauna and Suresh V. AmbudkarDagger

From the Laboratory of Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 14, 2000, and in revised form, January 10, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomes vis à vis the substrate, they show comparable t1/2 for both incorporation and release of nucleotide, and the affinities for [alpha -32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [alpha -32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of the plasma membrane protein, P-glycoprotein (Pgp),1 confers multidrug resistance to tumor cells. It belongs to the ATP-binding cassette (ABC) superfamily of transport proteins, characterized by two homologous halves containing six transmembrane helices and one nucleotide-binding site in each half (1, 2). Pgp confers drug resistance to tumor cells by extruding cytotoxic natural product hydrophobic drugs using the energy of ATP hydrolysis (2, 3). Pgp interacts not only with nucleotides (4-6) and cytotoxic drugs (3) but also with a diverse set of other lipophilic compounds (7). Additionally, besides the transport of these compounds coupled to ATP hydrolysis, other complex interactions are known to occur in Pgp with modulators of the multidrug resistance (MDR) phenotype (8). Considering the importance of Pgp in cancer chemotherapy and as a model system for ABC transporters in general, a clear understanding of the catalytic cycle of this transporter is of considerable importance.

The first catalytic scheme proposed for Pgp (9) was that of the ATP hydrolysis reaction. The essential feature of this model is alternating hydrolysis of ATP at the two ATP-binding sites. It was postulated that nucleotide first binds to one of the two sites but could not be hydrolyzed. When another nucleotide binds to the second site it promotes hydrolysis at the first site, which in turn powers substrate transport. In the next cycle, hydrolysis occurs at the second ATP site. We have recently (10) demonstrated that there is substantially greater complexity in the catalytic cycle of Pgp. We simultaneously monitored changes in the substrate- and nucleotide-binding sites to show that although binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of the substrate [125I]iodoarylazidoprazosin ([125I]IAAP) for Pgp trapped in the transition state conformation (Pgp·ADP·Vi) is reduced >30-fold. Even more remarkable is the finding that to transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e. a conformation that binds substrate with high affinity, the hydrolysis of an additional molecule of nucleotide is obligatory. This requirement for two ATP hydrolysis events in the course of a single catalytic cycle was consistent with the measured stoichiometry for Pgp where two ATP molecules are hydrolyzed for each substrate molecule transported (11, 12).

In this study we have analyzed, in real time, the repeating succession of vanadate (Vi)-induced trapping and release of [alpha -32P]8-azido-ADP through an entire catalytic cycle, and we monitored the substrate ([125I]IAAP) binding at the beginning and end of each trapping (hydrolysis) event. The results, besides validating our model, provided experimental strategies for independently studying the kinetics of the two hydrolysis events. We find that at the end of the first hydrolysis event there is a >30-fold decrease in the affinity of nucleotide for Pgp coincident with the decreased substrate binding shown earlier (10, 13). Release of occluded ADP or 8-azido-ADP is adequate for the next hydrolysis event to occur but is not sufficient for recovery of substrate binding, which occurs only at the end of the second hydrolysis, i.e. after ADP dissociates from the transporter. Whereas the two hydrolysis events have different functional outcomes vis à vis the substrate, they show comparable t1/2 for both incorporation and release of nucleotide, and the Km values for [alpha -32P]8-azido-ATP during the Vi-induced trapping during both hydrolysis events are identical. Similarly, the incorporation of [alpha -32P]8-azido-ADP in both the N-terminal and the C-terminal ATP sites is identical during both ATP hydrolysis events. These data demonstrate that during individual hydrolysis events the ATP sites are recruited in a random manner, and the fact that only one site is utilized at any given time is a consequence of the conformational change that reduces the affinity of nucleotide for the second ATP site. Thus, the prevention of ATP binding to the second site while the first one is in a catalytic conformation appears to be the explanation for alternate catalysis in Pgp.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Cyclosporin A was purchased from Calbiochem. and [125I]Iodoarylazidoprazosin ([125I]IAAP) (2, 200 Ci/mmol) was obtained from PerkinElmer Life Sciences. [alpha -32P]8-Azido-ATP (15-20 Ci/mmol) and 8-azido-ATP were purchased from Affinity Labeling Technologies, Inc (Lexington, KY). TNP-ATP was procured from Molecular Probes (Eugene OR). All other chemicals were obtained from Sigma.

Preparation of Crude Membranes from High Five Insect Cells Infected with Recombinant Baculovirus Carrying the Human MDR1 Gene-- High Five insect cells (Invitrogen, Carlsbad, CA) were infected with the recombinant baculovirus carrying the human MDR1 cDNA with a His6 tag at the C-terminal end (BV-MDR1 (H6)) as described (13). Crude membranes were prepared as described previously (4, 13).

Purification of Pgp-- Human Pgp was purified as described previously (13) with minor changes. Briefly, crude Pgp-containing membranes were solubilized with octyl-beta -D-glucopyranoside (1.25%) in the presence of 20% glycerol and lipid mixture (0.1%). Solubilized proteins were subjected to metal affinity chromatography (Talon resin, CLONTECH, Palo Alto, CA) in the presence of 0.95% octyl-beta -D-glucopyranoside and 0.04% lipid; 80% purified Pgp was eluted with 100 mM imidazole. Pgp in 100 mM imidazole fraction was then concentrated (Centriprep-50, Amicon, Beverly, MA) to ~0.5 mg/ml and stored at -70 °C. Pgp was identified by immunoblot analysis using the monoclonal antibody C219 (13) and quantified by Amido Black protein estimation method as described previously (14).

Photoaffinity Labeling of Pgp with [125I]IAAP-- The crude membranes (10-50 µg) were incubated with the drug or modulator for 3 min at room temperature in 50 mM Tris-HCl, pH 7.5, and [125I]IAAP (unless otherwise stated, 3-6 nM) was added and incubated for an additional 5 min under subdued light. The samples were then illuminated with a UV lamp assembly (PGC Scientifics, Gaithersburg, MD) fitted with two black light (self-filtering) UV-A long wave F15T8BLB tubes (365 nm) for 10 min at room temperature (21-23 °C). Following SDS-PAGE on a 8% Tris glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film (Eastman Kodak Co.) at -70 °C for 12-24 h. The radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT.

Vanadate-induced [alpha -32P]8-Azido-ADP Trapping in Pgp-- The Pgp·ADP·Vi or Pgp·8-azido-ADP·Vi transition state conformation was generated as described previously with minor modifications (10). Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer (40 mM MES-Tris, pH 6.8, 50 mM KCl, 5 mM sodium azide, 2 mM EGTA, 2 mM dithiothreitol, and 10 mM MgCl2) containing 50 µM [alpha -32P]8-azido-ATP (containing 4 µCi/nmol) and 250 µM Vi in the dark at 37 °C for 5 min. The reaction was stopped by the addition of 12.5 mM ice-cold ATP and placing the sample immediately on ice. The trapped nucleotides were photo-cross-linked as described below.

Binding of [alpha -32P]8-Azido-ATP to Pgp-- Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer containing 10 µM [alpha -32P]8-azido-ATP (containing 2-5 µCi/nmol) in the dark at 4 °C for 5 min. The samples were then irradiated with an UV lamp assembly (365 nm) for 10 min on ice (4 °C). Ice-cold ATP (12.5 mM) was added to displace excess noncovalently bound [alpha -32P]8-azido-ATP. Excess nucleotides were removed by centrifugation at 300,000 × g at 4 °C for 10 min by using S120-AT2 rotor in a RC-M120EX micro-ultracentrifuge (Sorvall, Newtown, CT), and the pellet was resuspended in 1× SDS-PAGE sample buffer. Following SDS-PAGE on an 8% Tris glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film at -70 °C for 12-24 h. The radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT.

Binding of TNP-ATP to Pgp-- Binding of the fluorescent analogue TNP-ATP was measured by determining the increase in fluorescence signal of TNP-ATP in the presence of purified Pgp incorporated into proteoliposomes. Proteoliposomes (20-25 µg of protein) were incubated with 100 µM TNP-ATP in ATPase assay buffer in a reaction volume of 200 µl for 10 min at room temperature (22-23 °C) under subdued light. The samples were then transferred to individual wells in a 96-well flat bottom, UV-transparent, disposable microplate (Spectraplate, Molecular Devices, Sunnyvale, CA) and an emission scan (500-600 nm) obtained using excitation at 408 nm on a Spectra MAX 250 Microplate spectrofluorimeter (Molecular Devices, Sunnyvale, CA). The enhancement in fluorescence due to TNP-ATP binding was estimated by running an emission scan of TNP-ATP in the assay buffer in the absence of Pgp. Additionally, to obtain signal specific to Pgp, an equivalent emission scan in the presence of 10 mM ATP (i.e. 100-fold excess) was generated and subtracted from the scan obtained in the absence of ATP.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Vi-induced ADP-trapped Conformation of Pgp during ATP Hydrolysis Exhibits a Marked Decrease in Affinity for Both Drug Substrate [125I]IAAP and [alpha -32P]8-Azido-ATP-- 8-Azido-ATP is a good hydrolysis substrate for both Chinese hamster and human Pgp, with a Km similar to ATP (6, 15, 16). alpha -32P-Labeled 8-azido-ATP is a useful photoaffinity reagent to understand the nucleotide binding and ATP hydrolysis events that accompany drug-substrate transport in Pgp. Our recent studies (10) show that binding of nucleotide to Pgp per se does not affect its interactions with the substrate, similarly Fig. 1 demonstrates that the binding of the nucleotide, [alpha -32P]8-azido-ATP, is not affected by substrates such as verapamil and cyclosporin A. Earlier work from our laboratory (10, 13) demonstrated that the Vi-trapped conformation of Pgp binds substrates with at least 30-fold reduced affinity. The data in Fig. 2A show that if the photoaffinity substrate analogue, [125I]IAAP, is allowed to bind Pgp, which has been treated previously with increasing concentrations of 8-azido-ATP in the presence of Vi at 37 °C (which corresponds to increased trapping of 8-azido-ADP in Pgp), there is a concentration-dependent decrease in substrate binding. A single phase exponential decay model fits the data with a Ki = 18 µM for the 8-azido-ATP inhibition of [125I]IAAP binding. It is also clear from these data that for maximal trapping of 8-azido-ADP in the presence of Vi, 0.5 to 1 mM 8-azido-ATP is required. Our earlier work (10) and data in Fig. 2A clearly show that the Pgp·8-azido-ADP·Vi conformation of Pgp exhibits a large decrease in affinity for substrate. This raises the question as to how nucleotide binding to Pgp is affected in the Vi-trapped state (i.e. transition state). To address this question we generated the Pgp·8-azido-ADP·Vi complex in the presence of increasing concentrations of 8-azido-ATP and Vi, washed off excess nucleotide and Vi by centrifugation, and then quantified the extent of [alpha -32P]8-azido-ATP binding to Pgp. The results of this experiment are graphically depicted in Fig. 2B. The Vi-induced 8-azido-ADP-trapped conformation of Pgp shows a concentration-dependent decrease in binding of nucleotide, similar to that seen for substrate (Fig. 2A). A single phase exponential decay model fits the data, and the Ki of 8-azido-ATP (during trapping) for the inhibition of nucleotide binding is 12 µM and is comparable to the Ki of 8-azido-ATP (during trapping) for the inhibition of [125I]IAAP binding which is 18 µM.



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Fig. 1.   Effect of substrates on the binding of [alpha -32P]8-azido-ATP to Pgp. Crude membranes (20 µg of protein) were incubated in the ATPase assay buffer with Me2SO, 50 µM verapamil, or 10 µM cyclosporin A at 37 °C for 10 min. The samples were transferred to ice, and 10 µM [alpha -32P]8-azido-ATP (containing 4 µCi/nmol) was added under subdued lighting, incubated at 4 °C for 5 min, and then UV-irradiated (365 nm) for 10 min on ice (4 °C). Ice-cold ATP (12.5 mM) was added to displace noncovalently bound [alpha -32P]8-azido-ATP, and excess nucleotides were removed by centrifugation at 300,000 × g at 4 °C for 10 min. The pellet was resuspended in 1× SDS-PAGE sample buffer, and following SDS-PAGE the gels were dried and exposed to x-ray film at -70 °C for 12-24 h. Autoradiogram shows untreated Pgp (lane 1); Pgp pretreated with 50 µM verapamil (lane 2), or 10 µM cyclosporin A (lane 3).



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Fig. 2.   The Pgp·8-azido-ADP·Vi transition state complex shows reduced affinity for both [125I]IAAP and [alpha - 32P]8-azido-ATP. A, [125I]IAAP binding to Pgp pretreated with 8-azido-ATP and Vi. Crude membranes (20 µg of protein) were treated with increasing concentrations of 8-azido-ATP (1-1000 µM) and Vi (250 µM) at 37 °C for 10 min in the dark. Excess 8-azido-ATP and Vi were removed by centrifugation at 300,000 × g at 4 °C for 10 min. The membranes were resuspended in 50 mM Tris-HCl, pH 7.5, and labeled with 5 nM [125I]IAAP as described under "Experimental Procedures." [125I]IAAP incorporated in the Pgp band was quantified using a PhosphorImager. B, [alpha -32P]8-azido-ATP binding to Pgp pretreated with 8-azido-ATP and Vi. Crude membranes (20 µg of protein) were treated with increasing concentrations of 8-azido-ATP and Vi as described in A. Excess 8-azido-ATP and Vi were removed by centrifugation at 300,000 × g at 4 °C for 10 min. The membranes were resuspended in ATPase assay buffer and labeled with 10 µM [alpha -32P]8-azido-ATP (4 µCi/nmol) at 4 °C, as described in "Experimental Procedures." [alpha -32P]8-Azido-ATP incorporated in the Pgp band was quantified using a PhosphorImager. Inset shows the Pgp band labeled with [alpha -32P]8-azido-ATP that has been quantified in the graph. C, determination of the affinity of Pgp for [alpha -32P]8-azido-ATP before and after Vi-induced 8-azido-ADP trapping. Crude membranes (20 µg of protein) were incubated with increasing concentrations of [alpha -32P]8-azido-ATP (5-10 µCi/nmol) at 4 °C, as described under "Experimental Procedures." Excess nucleotide was removed by centrifugation at 300,000 × g at 4 °C for 10 min. The membranes were resuspended in 1× SDS-PAGE buffer and electrophoresed on an 8% Tris glycine gel. [alpha -32P]8-Azido-ATP incorporated in the Pgp band was quantified using a PhosphorImager. Figure shows binding of [alpha -32P]8-azido-ATP to control () membranes and those pretreated with 1.25 mM 8-azido-ATP and 250 µM Vi at 37 °C for 10 min as described in B to trap Pgp in the transition state conformation (black-triangle).

To quantify the decrease in affinity for nucleotide in the Pgp·8-azido-ADP·Vi complex, we estimated the apparent Kd value of 8-azido-ATP for Pgp before and after Vi-induced trapping. Fig. 2C depicts that [alpha -32P]8-azido-ATP binding to control membrane vesicles shows saturation; a hyperbola fits the data suggesting a single binding site or more than one site with the same affinity, and the Kd of [alpha -32P]8-azido-ATP was computed to be 13 µM. Pgp previously trapped with Vi (250 µM) and 8-azido-ATP (1.2 mM) on the other hand shows greatly reduced binding of [alpha -32P]8-azido-ATP, which is not saturable even at a concentration of 100 µM (data not shown). This suggests a large decrease (>30-fold) in the affinity of nucleotide for Pgp in the Vi-trapped conformation.

The Vi-induced ADP-trapped Conformation of Pgp during ATP Hydrolysis Exhibits a Marked Decrease in Affinity for the Fluorescent Nucleotide TNP-ATP-- To exclude the possibility that the reduced binding of nucleotide to Pgp in the transition state is an artifact of photoaffinity cross-linking, another approach using the fluorescent nucleotide analogue TNP-ATP was used. TNP-ATP is a hydrolyzable, fluorescent analogue of ATP (17). Both Chinese hamster and human Pgp can hydrolyze TNP-ATP but at a much slower rate than ATP, and TNP-ATP has also been used to characterize the ATP sites on Pgp (18, 19). The compound exhibits a low quantum yield in an aqueous medium with an emission maxima at 550 nm (excitation 408 nm), and an increase in quantum yield accompanied by a small blue shift (new emission maxima at 530 nm) occurs when TNP-ATP binds to the ATP sites of Pgp (19). However, when Pgp is pretreated with Vi and 8-azido-ATP at 37 °C to generate the Pgp·8-azido-ADP·Vi complex, there is a marked decrease in the fluorescence (Fig. 3A) suggesting that the binding of nucleotide (TNP-ATP) to Pgp is drastically reduced, consistent with the results obtained in the experiments with [alpha -32P]8-azido-ATP (Fig. 2, B and C).



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Fig. 3.   The Pgp·8-azido-ADP·Vi transition state complex shows reduced binding of the fluorescent nucleotide TNP-ATP. A, TNP-ATP binding to Pgp in transition state conformation generated by pretreatment with 8-azido-ATP and Vi. Purified Pgp was reconstituted into liposomes as described previously (16). Proteoliposomes (25 µg of protein) were incubated with 100 µM TNP-ATP at room temperature for 10 min, and emission scans were obtained (excitation = 408 nm; emission = 500-600 nm). Scans taken in the presence of Pgp were corrected for nonspecific binding by subtracting scans acquired in the presence of 100-fold excess ATP. Figure shows TNP-ATP + Pgp, control (continuous line) and TNP-ATP + Pgp pre-trapped with 8-azido-ADP and Vi (dashed line). Pgp was trapped with 8-azido-ADP by incubating with 8-azido-ATP, 1.25 mM in the presence of Vi, 0.25 mM for 10 min at 37 °C. The reaction was stopped by the addition of 10 mM ice-cold ATP, and excess nucleotides and Vi were removed by centrifugation. B, kinetics of inhibition of TNP-ATP binding to Pgp pretreated with ATP and Vi. Proteoliposomes containing purified Pgp (20-25 µg) were treated with increasing concentrations of ATP and Vi (250 µM) at 37 °C for 10 min. Excess ATP and Vi were removed by centrifugation at 300,000 × g at 4 °C for 10 min. The membranes were resuspended in ATPase assay buffer and incubated with 100 µM TNP-ATP at room temperature for 10 min, and emission scans were obtained (excitation = 408 nm; emission = 500-600 nm). Scans taken in the presence of Pgp were corrected for nonspecific binding by subtracting scans acquired in the presence of 100-fold excess ATP. The peak fluorescence signal at 530 nm for TNP-ATP at each concentration of ATP in the presence of Vi was determined, and the data were normalized to the fluorescence of the sample with no ATP. The data were fitted using GraphPad Prism 2.0 for the PowerPC Macintosh.

The kinetics of inhibition of TNP-ATP binding to Pgp in the presence of ATP and Vi are depicted in Fig. 3B. Purified, reconstituted Pgp was treated with increasing concentrations of ATP in the presence of Vi at 37 °C for 10 min, and excess ATP and Vi were removed by centrifugation. Extent of TNP-ATP binding to Pgp was quantified by measuring the fluorescence intensity (Ex = 408 nm and Em = 530 nm). A single phase exponential decay model fits the data with a Ki(ATP) of 83 µM.

Dissociation of [alpha -32P]8-Azido-ADP from the Pgp·8-Azido-ADP·Vi Complex Is Not Affected by Nucleotides-- The results thus far show that following ATP hydrolysis, affinity for nucleotide is considerably reduced. There is considerable evidence in the literature that there is communication between the two ATP sites of Pgp and that mutations or chemical modification of residues in either ATP site precludes ATP hydrolysis (3, 6, 15, 20-25). This has led to the hypothesis that there is alternating hydrolysis of ATP at the two ATP sites (9, 26, 27), i.e. that nucleotide first binds to one of the two sites but cannot be hydrolyzed. When another nucleotide binds to the second ATP site it promotes hydrolysis at the first site, which in turn powers substrate transport. In the context of our results this raises the following two questions. (a) Does nucleotide binding per se affect Vi-induced trapping? (b) Once ATP hydrolysis ensues, do nucleotides influence the release of ADP from Pgp? To address the first question, we monitored Vi-induced trapping of [alpha -32P]8-azido-ADP in the presence increasing concentrations of AMPPNP, a nucleotide analogue, that can bind to Pgp but cannot be hydrolyzed (10). Thus, if binding of nucleotide at one ATP site exerts allosteric control over hydrolysis at the other ATP site, binding of AMPPNP at one site should stimulate Vi-induced trapping at the other site. Fig. 4A, however, clearly demonstrates that AMPPNP inhibits in a dose- dependent manner the Vi-induced trapping of [alpha -32P]8-azido-ADP with an apparent Ki = 1.3 mM. This suggests that binding of nucleotide at one site does not stimulate ATP hydrolysis at the other site.



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Fig. 4.   Effect of nucleotides on the Vi-induced trapping of [alpha -32P]8-azido-ADP and on the dissociation of [alpha -32P]8-azido-ADP from the Pgp·[alpha -32P]8-azido-ADP·Vi complex. A, effect of the nonhydrolyzable nucleotide AMPPNP on Vi-induced trapping of [alpha -32P]8-azido-ADP into Pgp. Crude membranes (1 mg/ml protein) were incubated in the dark at 37 °C for 10 min with 50 µM [alpha -32P]8-azido-ATP (3-5 µCi/nmol), 250 µM Vi, and increasing concentrations of AMPPNP in the ATPase assay buffer. The reaction was stopped by adding 12.5 mM ice-cold ATP and placing the tubes on ice. Following photo-cross-linking by UV irradiation at 365 nm for 5 min and SDS-PAGE, the level of [alpha -32P]8-azido-ADP incorporated was quantified using a PhosphorImager, and the data were fit by using GraphPad Prism 2.0 for the PowerPC Macintosh. A single phase exponential decay model best fits the data. B, nucleotides have no effect on the rate of dissociation of [alpha -32P]8-azido-ADP from the Pgp·8-azido-ADP·Vi complex. Crude membranes (1 mg/ml protein) were incubated in the dark at 37 °C for 10 min with 50 µM [alpha -32P]8-azido-ATP (3-5 µCi/nmol) and 250 µM Vi in the ATPase assay buffer. The reaction was stopped by adding 12.5 mM ice-cold ATP and placing the tubes on ice. Untrapped nucleotides and excess Vi were removed by centrifugation at 300,000 × g for 10 min, and the membranes were resuspended in the Mg2+-free ATPase assay buffer and divided into 4 aliquots. To these were added 10 mM MgCl2, control membranes; 10 mM MgCl2 + 1.25 mM ATP; 10 mM MgCl2 + 1.25 mM AMPPNP; or 10 mM EDTA, respectively, and incubated at 37 °C. Aliquots were removed at indicated intervals and placed on ice and photo-cross-linked by UV irradiation at 365 nm for 5 min, and SDS-PAGE was performed on each sample. The level of [alpha -32P]8-azido-ADP incorporated was quantified using a PhosphorImager, and the data were fit using GraphPad Prism 2.0 for the PowerPC Macintosh. Dissociation of [alpha -32P]8-azido-ADP in control membranes () and in the presence of 1.25 mM ATP (black-down-triangle ), 1.25 mM AMPPNP (black-square), or in Mg2+-free medium containing 10 mM EDTA (black-triangle) are shown in the figure.

To address the second question, i.e. what controls the release of ADP from Pgp after ATP hydrolysis, we monitored the dissociation of [alpha -32P]8-azido-ADP from the Pgp·8-azido-ADP·Vi complex. The [alpha -32P]8-azido-ADP was trapped by incubating crude membranes with [alpha -32P]8-azido-ATP and Vi at 37 °C for 10 min, and free Vi and nucleotide were removed by centrifugation, and the resuspended membranes were incubated at 37 °C under different conditions. Aliquots were removed at intervals, and excess cold ATP was added to displace the [alpha -32P]8-azido-ADP dissociated from the complex, and the samples were cross-linked by UV irradiation. Fig. 4B shows that the [alpha -32P]8-azido-ADP is completely dissociated within 15 min at 37 °C with a t1/2 of 2-3 min. The t1/2 for dissociation is not influenced by the presence of excess 8-azido-ADP, 8-azido-ATP, ATP, or the nonhydrolyzable nucleotide AMPPNP, added in the presence of Mg2+. The dissociation is, however, strongly temperature-dependent with no appreciable dissociation during a 15-min incubation at room temperature (data not shown). These results suggest that following ATP hydrolysis, ADP dissociates spontaneously and that while in the transition state, ADP or 8-azido-ADP is occluded and not exchangeable with exogenously added nucleotide.

Characterization of the Two ATP Hydrolysis Events in a Single Catalytic Cycle of Pgp-- The experiments described above demonstrate that the ATP hydrolysis that follows binding of nucleotide and substrate results in a conformational change in Pgp that dramatically lowers the affinity of both substrate and nucleotide and inhibits the binding of additional molecules. Fig. 5A depicts an experimental strategy, described in detail in the legend, to discriminate between the two hydrolysis events postulated to occur in a single catalytic cycle of Pgp (10). Essentially, Pgp can be put through repeating cycles of Vi-induced [alpha -32P]8-azido-ADP trapping and release, and the kinetic parameters for each step can be determined. In Fig. 5B we depict the rate constants for the repeating cycles of Vi-induced [alpha -32P]8-azido-ADP trapping and release. The data show that the rate constants for two hydrolysis events (cf. Fig. 5B) are indistinguishable. The occurrence of two ATP hydrolysis events in a single catalytic cycle raises the question, is there a functional asymmetry between the two ATP sites? For example, is the N-terminal ATP site committed to the first hydrolysis event followed by the recruitment of the C-terminal ATP site for the second ATP hydrolysis event? We addressed this question by performing a mild trypsin digestion of Pgp after labeling with [alpha -32P]8-azido-ADP at steps II, IV, and VI of the catalytic cycle, as depicted in Fig. 5A. The results (Fig. 5C) clearly demonstrate that at high concentration of [alpha -32P]8-azido-ATP, there is no preference for either the N- or the C-terminal ATP site during any of the ATP hydrolysis events.



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Fig. 5.   Repeating cycles of ATP hydrolysis. A, schematic representation of the experiment. Step I and II, crude membranes (protein, 1 mg/ml) were incubated in the dark at 37 °C with 50 µM [alpha -32P]8-azido-ATP (3-5 µCi/nmol) and 250 µM Vi in the ATPase assay buffer. Aliquots were removed at regular intervals over 15 min and placed on ice. The reaction was stopped by adding excess ice-cold ATP (12.5 mM) and photo-cross-linking by UV irradiation at 365 nm for 5 min on ice. Steps II and III, at the end of 15 min, the reaction mixture was transferred to ice, and un-trapped nucleotides and excess Vi were removed by centrifugation at 300,000 × g for 10 min. The membranes were resuspended in the ATPase assay buffer and placed at 37 °C. Aliquots were again removed at indicated intervals over the next 20 min (35 min from start of experiment), and photo-cross-linked as described above. Step III and IV, at the end of 35 min, fresh 50 µM [alpha -32P]8-azido-ATP and 0.25 mM Vi were added to initiate the next cycle of ATP hydrolysis. Aliquots were removed at regular intervals, over an additional 15 min (50 min from start of experiment) and photo-cross-linked as described above. At the end of 15 min the reaction mixture was transferred to ice, and un-trapped nucleotides and excess Vi were removed by centrifugation at 300,000 × g for 10 min, and the membranes were resuspended in the ATPase assay buffer and placed at 37 °C. Steps IV and V, the next cycle of dissociation of [alpha -32P]8-azido-ADP was monitored by placing the reaction mixture at 37 °C and removing aliquots as indicated above. Steps V and VI, at the end of 20 min (70 min from start of experiment) a third hydrolysis cycle was initiated by adding 50 µM [alpha -32P]8-azido-ATP and 0.25 mM Vi and followed over the next 10 min (80 min from start of experiment). At the end of the experiment, all samples were electrophoresed and the [alpha -32P]8-azido-ADP incorporated into the Pgp band quantified using a PhosphorImager. The data were fit using GraphPad Prism 2.0 for the Power Macintosh (see below). The thick vertical arrows represent additions of [alpha -32P]8-azido-ATP and Vi at steps I, III, and V. B, the cycles of [alpha -32P]8-azido-ADP trapping and release. The individual fits are for the experiment described in A above. Initiation of the first hydrolysis event (panels I and II), dissociation of [alpha -32P]8-azido-ADP following the first hydrolysis event (panels II and III), initiation of the second hydrolysis event (panels III and IV), dissociation of [alpha -32P]8-azido-ADP following the second hydrolysis event (panels IV and V), and initiation of the third hydrolysis event, i.e. initiation of the next catalytic cycle (panels V and VI). The data were fitted to a single phase exponential decay model and the t1/2 was determined for each step. C, distribution of Vi-trapped [alpha -32P]8-azido-ADP in the two ATP sites. At steps II, IV, and VI (described in Fig. 4A), following photo-cross-linking, the samples were treated with trypsin (protein:trypsin, 10:1) for 5 min at 37 °C to separate N- and C-terminal halves of Pgp (8). The reaction was stopped by the addition of 5-fold excess soybean trypsin inhibitor, and SDS-PAGE samples in the presence of 5 M urea were prepared by incubation at 23 °C for 20 min. Following SDS-PAGE, radioactivity in the N- and the C-terminal fragments in a dried gel were quantified using a PhosphorImager. Figure shows distribution of [alpha -32P]8-azido-ADP in the N- (filled bars) and C-terminal (open bars) halves of Pgp at steps II, IV, and VI as depicted in A above. The values represent an average of 3 independent experiments, and the standard deviations are shown by error bars.

Binding of [125I]IAAP to Pgp at Various Steps in the Catalytic Cycle of ATP Hydrolysis-- We postulated earlier that two hydrolysis events occur during a single cycle of Pgp (10) based on the following evidence: (a) following ATP hydrolysis Pgp takes on a conformation with >30-fold reduced affinity for substrate, and (b) for Pgp to regain the conformation that binds substrate with high affinity a second ATP hydrolysis event is mandatory. Although Fig. 5B tracks ATP hydrolysis over the entire catalytic cycle and is consistent with our previous findings, it does not explicitly demonstrate the status of the substrate-binding site prior to and following each hydrolysis event. Fig. 6 shows [125I]IAAP binding to Pgp at different steps in the catalytic cycle as depicted in Fig. 5B, which, re-plotted on a continuous time scale, is shown as a gray backdrop. The gray area thus represents the extent of [alpha -32P]8-azido-ADP incorporation and release in a single catalytic cycle, and the filled bars depict the extent of [125I]IAAP incorporated into Pgp at various steps in the catalytic cycle (see Fig. 5, A and B). The data for [alpha -32P]8-azido-ADP incorporation and release, which have been quantitatively treated in Fig. 5B, merely offer a visual representation in Fig. 6 of the state of Pgp molecules in a single catalytic cycle.



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Fig. 6.   Binding of [125I]IAAP to Pgp during various steps of the catalytic cycle. Crude membranes (20 µg of protein) were labeled with 5 nM [125I]IAAP at steps I-V in the catalytic cycle of Pgp as depicted in Fig. 4A. The extent of [125I]IAAP binding is superimposed on a representation of Fig. 5B in a continuous time scale (shaded gray area). The membranes were labeled with [125I]IAAP after the following pretreatments, Step I, control untreated membranes. Step II, membranes treated with 1.2 mM 8-azido-ATP and 250 µM Vi for 10 min at 37 °C. Step III, membranes at step II centrifuged to remove excess 8-azido-ATP and Vi, resuspended in ATPase buffer, and incubated for 15 min at 37 °C. Step IV, membranes at the end of step III were again incubated with 1.2 mM 8-azido-ATP and 250 µM Vi for 10 min at 37 °C. Step V, membranes in step IV were centrifuged to remove excess 8-azido-ATP and Vi, resuspended in ATPase buffer, and incubated for 15 min at 37 °C. Step Va depicts an aliquot of membranes at step III treated with 1.2 mM 8-azido-ATP in the absence of Vi for 10 min at 37 °C.

Prior to the first hydrolysis event corresponding to the zero time point in Fig. 5A (I), there is a normal level of [125I]IAAP binding (Fig. 6, step I), similar to untreated Pgp. Immediately following ATP hydrolysis, i.e. the final time point in Fig. 5A (step I) or the first time point in Fig. 5A (step II), the [125I]IAAP binding is reduced by >90% (Fig. 6, step II). Following release of occluded 8-azido-ADP, in the absence of ATP hydrolysis (the final time point in Fig. 5A (step II)), [125I]IAAP binding continues to be reduced (Fig. 6, step III). Vi-induced trapping of 8-azido-ADP in the second cycle (Fig. 5A (step III)) does not show any recovery of [125I]IAAP binding (Fig. 6, step IV). However, instead of trapping 8-azido-ADP, if hydrolysis is allowed to occur in the absence of Vi, there is full recovery of [125I]IAAP binding (Fig. 6, step Va). Similarly, release of the occluded 8-azido-ADP following the second hydrolysis (Fig. 5A (step V)) allows recovery of [125I]IAAP binding (Fig. 6, step V). Taken together with Fig. 5B, these results further support the model we have previously proposed (10) and establish that two kinetically similar but functionally different ATP hydrolysis events are occurring in a single catalytic cycle.

The Two Hydrolysis Events in a Single Catalytic Cycle of Pgp Have Identical Km Values for [alpha -32P]8-azido-ATP in the Presence of Vi-- To obtain the Km of [alpha -32P]8-azido-ATP for Pgp during Vi-induced trapping, crude membranes containing Pgp were incubated with increasing concentrations of [alpha -32P]8-azido-ATP in the presence of Vi at 37 °C. The reaction was stopped with excess ATP. [alpha -32P]8-Azido-ADP incorporated into Pgp was quantified from a SDS-PAGE gel as described under "Experimental Procedures." The extent of [alpha -32P]8-azido-ADP trapping in the first hydrolysis event is depicted in Fig. 7A. The single site model of Henri-Michaelis-Menten best described the data to give a Km of 18.4 µM. To determine the kinetics of the second hydrolysis event, the crude membranes were first allowed to trap 8-azido-ADP in the presence of Vi for 10 min; excess 8-azido-ATP and Vi were removed by centrifugation, and then the occluded 8-azido-ADP was allowed to disassociate by incubation at 37 °C for 15 min (steps I and II in Fig. 5A). The second hydrolysis event was followed in the presence of increasing concentrations of [alpha -32P]8-azido-ATP and Vi, and the extent of [alpha -32P]8-azido-ADP incorporation was quantified as described above. The data (Fig. 7B) show that the Km value for [alpha -32P]8-azido-ATP in the presence of Vi during the second hydrolysis event, 19.4 µM, is indistinguishable from that for the first hydrolysis event. However, the results depicted in Fig. 7 substantiate the notion that although the hydrolysis events measured in Fig. 7, A and B, are kinetically similar, they occur in two distinct conformations of Pgp vis à vis the substrate binding (Figs. 5B and 6).



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Fig. 7.   Determination of the Km of [alpha -32P]8-azido-ATP in the presence of Vi-induced trapping during two ATP hydrolysis events in a single catalytic cycle of Pgp. A, the Km of [alpha -32P]8-azido-ATP during the first hydrolysis event. Crude membranes were incubated with increasing concentrations of [alpha -32P]8-azido-ATP (3-5 µCi/nmol) in the presence of 250 µM Vi at 37 °C; at the end of 10 min the reaction was stopped with excess ATP. [alpha -32P]8-Azido-ADP incorporated into Pgp was quantified from a SDS-PAGE gel as described under "Experimental Procedures." B, the Km of [alpha -32P]8-azido-ATP during the second hydrolysis event. To study the kinetics of the second hydrolysis event, the crude membranes were first allowed to trap 8-azido-ADP by incubating with 1.25 mM 8-azido-ATP in the presence of 250 µM Vi for 10 min, and excess 8-azido-ATP and Vi were removed by centrifugation, and then the occluded 8-azido-ADP was allowed to dissociate by incubation at 37 °C for 15 min. These membranes were then incubated with increasing concentrations of [alpha -32P]8-azido-ATP in the presence of 250 µM Vi at 37 °C in the ATPase assay buffer. At the end of 10 min, the reaction was stopped with excess ATP, and the incorporation of radiolabel into Pgp was quantified as described above. The data were analyzed using GraphPad Prism 2.0.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our recent work has demonstrated that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study, we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We also track, in real time, the repeating succession of trapping and release of [alpha -32P]8-azido-ADP through an entire catalytic cycle, and we independently study the kinetics of the two hydrolysis events. Crude membrane fractions as well as purified Pgp reconstituted into lipid vesicles manifest a basal level of ATP hydrolysis that is stimulated by substrates and modulators of Pgp by a factor of up to 10 (3, 9). Pgp exhibits low affinity for ATP (Km 0.3 to 1 mM) compared with, for example, myosin or the mitochondrial F1F0-ATP synthase (28, 29). Additionally, in Pgp no covalent phosphorylated (E-P) intermediate has been demonstrated as is known to occur for the P-type ATPases (9). These facts led Senior and co-workers (9, 26) to hypothesize that during ATP hydrolysis a state of high chemical potential is generated and that the relaxation of such a state powers the extrusion of substrate. Recent work from our laboratory (10) experimentally demonstrated a large conformational change accompanying ATP hydrolysis. We showed that the long lived Pgp·8-azido-ADP·Vi transition state complex, which is generated immediately following ATP hydrolysis, exhibits a drastic decrease in the affinity for the substrate analogue [125I]IAAP. Even more interesting was the observation that the release of the occluded 8-azido-ADP was not sufficient to regain substrate binding, which occurred only after an additional ATP hydrolysis step. That two hydrolysis events occur in a single catalytic cycle, one associated with efflux of drug and the other to bring about conformational changes that "reset" the molecule, raise several mechanistic questions about ATP hydrolysis during the catalytic cycle of Pgp. Is there conformational coupling between the drug-binding sites and the ATP sites, and by extension what is the status of the drug and nucleotide binding at each step in the catalytic cycle? What effect, if any, does ATP hydrolysis have on subsequent nucleotide binding? Do the two ATP hydrolysis events show different kinetics?

Implicit in the model for the catalytic cycle proposed by Senior and others (24, 26, 27) is long range conformational coupling between the drug- and nucleotide-binding sites. Although direct evidence for such a relationship was lacking, interactions such as the stimulation of ATPase activity by drugs (4, 5, 30) have been accepted as evidence for the structural interactions that underlie such a coupling (26, 27). Our recent work (10) and Fig. 2A on the other hand quantitatively corroborate that large changes occur in the substrate-binding site as a consequence of ATP hydrolysis. It is critical, however, to demonstrate that these effects are not coincidental. A comparison of the Km for Vi-induced trapping of [alpha -32P]8-azido-ADP (Fig. 7A) with the Ki (8-azido-ATP) for inhibition of [125I]IAAP binding during trapping (Fig. 2A) shows the two values to be almost identical (Km, 22 µM; Ki, 18 µM), and a cause-effect relationship between the two is plausible. Moreover, the fact that Vi-induced trapping of 8-azido-ADP inhibits substrate ([125I]IAAP) binding in a concentration-dependent manner (Fig. 2A) provides stronger and more direct evidence for interactions between the substrate- and nucleotide-binding sites. That these are long range interactions resulting from conformational changes in the protein is underscored by the fact that drugs do not affect nucleotide binding per se (Fig. 1), nor do nucleotides affect substrate binding in the absence of ATP hydrolysis (10).

The consequences of ATP hydrolysis on substrate binding have been elucidated elsewhere (10) and are described briefly above. To understand the effect of ATP hydrolysis on the affinity of nucleotides for the ATP sites, we once more exploited the Vi-trapped, transition state complex (Pgp·ADP·Vi). Fig. 2C illustrates that if crude membranes containing Pgp are pretreated with 8-azido-ATP and Vi, they show reduced binding of [alpha -32P]8-azido-ATP. The Ki(8-azido-ATP) for inhibition of [alpha -32P]8-azido-ATP binding (12.3 µM) is comparable to the Km([alpha -32P]8-azido-ATP) for Vi-induced trapping of [alpha -32P]8-azido-ADP (22 µM) and the Ki (8-azido-ATP) for inhibition of [125I]IAAP binding (19 µM). These data suggest that the conformational changes that follow ATP hydrolysis reduce the affinity of both substrate and nucleotide for Pgp and the extent of decrease in the affinity of the nucleotide; [alpha -32P]8-azido-ATP (Figs. 2C and 3) is comparable to that demonstrated earlier for the substrate [125I]IAAP. The release of [alpha -32P]8-azido-ADP from the complex is spontaneous and not affected by nucleotide binding (Fig. 4A), and this is sufficient for the next ATP hydrolysis event to ensue (Fig. 5B). The conformational changes occurring in the ATP sites are thus different from those in the substrate site in that, except for the release of occluded ADP, there is no additional energetic requirement for resetting the nucleotide-binding pocket.

There is strong evidence to support the occurrence of two hydrolysis events in a single catalytic cycle. Useful information should be gained by studying these two events independently. Data depicted in Fig. 5B, which follows the experimental strategy depicted in Fig. 5A, show that it is experimentally possible to propel Pgp through repeating cycles of Vi-induced [alpha -32P]8-azido-ADP trapping and [alpha -32P]8-azido-ADP release. Although the t1/2 for each trapping and release event are comparable, Fig. 6 shows how the conformational state of the molecule differs during each of these events. It is clear from the gray-shaded area in Fig. 6 (data shown in Fig. 5B) that the two hydrolysis events are indistinguishable. Concurrent measurements of substrate binding, however, distinguish different states of the Pgp molecule in which these two events occur. Thus, when the first hydrolysis event is initiated (Fig. 6, step I), [125I]IAAP binding is not affected in contrast to over 90% inhibition of [125I]IAAP binding when the second hydrolysis event is initiated (Fig. 6, step III). At the end of the second hydrolysis event, however, [125I]IAAP binding is restored to normal levels (Fig. 6, step V) bringing Pgp to a state comparable to step I both in terms of substrate and nucleotide binding, suggesting the completion of one catalytic cycle. It would thus be reasonable to conclude that determining the kinetics of Vi-induced [alpha -32P]8-azido-ADP trapping between steps I and II, as depicted in Fig. 5, would represent the first hydrolysis event and between steps III and IV the second event. Fig. 7 shows that the Km ([alpha -32P]8-azido-ATP) for trapping is identical for the two hydrolysis events. Thus, even though these events have different functional consequences, they are kinetically indistinguishable. This would suggest that it is unlikely that the two hydrolysis events are individually associated with each ATP site. We propose that the nucleotide-binding site for any hydrolysis event at high nucleotide concentration is recruited randomly, that all hydrolysis events are kinetically equivalent, and that the different functional outcomes are a result of the conformational state of the Pgp molecule when a particular hydrolysis event occurs. This view is also consistent with the findings of Urbatsch et al. (31) that the two ATP sites are functionally equivalent. The fact that following Vi-induced trapping [alpha -32P]8-azido-ADP is distributed equally in both N- and C-terminal ATP sites (Fig. 5C) at steps II, IV, and VI, as depicted in Fig. 5A, further supports this view.

Earlier studies with plasma membranes from Chinese hamster ovary cells overexpressing Pgp clearly showed that the trapped [alpha -32P]8-azido-ADP labels the two ATP sites in equal proportion (32, 33). Our results with recombinant human Pgp similarly demonstrate that this distribution remains constant regardless of whether it is monitored at step II, IV, or VI of the cycle as depicted in Fig. 5A (Fig. 5C). These results strongly favor the conclusion that the two ATP sites are recruited randomly and show similar kinetics, which emerge as a single Km(ATP or 8-azido-ATP) during hydrolysis (6, 13, 15, 16). Moreover, the observation that trapping of 1 mol of ADP/mol of Pgp is sufficient to block ATP hydrolysis (34, 35) has been interpreted to mean that trapping of nucleotide at one site blocks catalysis at both sites. This observation has in turn led to the speculation (26, 27) that the binding of nucleotide at one ATP site is not sufficient for hydrolysis to occur and that the binding of nucleotide at the second ATP site permits hydrolysis at the first site by an allosteric mechanism. Data presented in this work, on the other hand, provide direct experimental evidence that following ATP hydrolysis the affinity of nucleotide for Pgp is drastically reduced (Figs. 2C and 3). Thus, the fact that trapping nucleotide at either ATP site blocks hydrolysis at both can be explained by this conformational change that drastically reduces the affinity of nucleotide for the second ATP site. The reduced affinity for nucleotides to Pgp in the transition state is also demonstrated by using the fluorescent nucleotide analogue, TNP-ATP (Fig. 3, A and B). Such a perspective would also be consistent with the characterization of ATP hydrolysis in Pgp (28) which shows Henri-Michaelis-Menten kinetics with a single Km in the 0.3-1 mM range, since no cooperativity has been demonstrated vis à vis the kinetics of ATP hydrolysis to suggest allosteric modulation. Finally, as depicted in Fig. 4A, binding of the nonhydrolyzable nucleotide AMPPNP does not stimulate Vi-induced trapping. If binding of nucleotide at one ATP site exerts a positive allosteric control over hydrolysis at the other, stimulation of hydrolysis should result in an increase in Vi-induced nucleotide trapping.

On the basis of this study, we propose a model for the catalytic cycle of Pgp, which is an extension of the one we proposed recently (10), and this is illustrated in Fig. 8. The drug and ATP first bind to Pgp (step I), because there is no energetic requirement for this step (36). Additionally, drugs do not affect nucleotide binding (Fig. 1) nor do nucleotides influence the binding of substrate. The binding of nucleotide and drug is followed by the first hydrolysis event (step II), which is accompanied by a conformational change that reduces the affinity of both substrate (Ref. 10 and Fig. 2A) and nucleotide (Figs. 2C and 3A) for Pgp. This intermediate can be trapped by using Vi, an analogue of Pi that generates the stable Pgp·ADP·Vi complex (step IIIA). Following hydrolysis, ADP is released (step IV). This release occurs spontaneously and is not influenced by the presence of nucleotides (Fig. 4B). The dissociation of ADP is accompanied by a conformational change that allows nucleotide binding (Fig. 5B) but substrate binding continues to be reduced (Fig. 6). A second ATP hydrolysis event is then initiated (step V) which is kinetically indistinguishable from the first (Fig. 7), at which point the substrate binding is still not regained (Fig. 6). This event too can be captured as an intermediate using Vi to trap the nucleotide (steps VIA and VIB). The subsequent release of ADP (step VII) completes one catalytic cycle bringing the Pgp molecule back to the original state where it can bind both substrate and nucleotide to initiate the next cycle (Fig. 6). This revised model thus incorporates key elements based on the work reported here to the scheme that we proposed earlier (10). The conformation of Pgp, following ATP hydrolysis, shows reduced affinity for the nucleotide (steps II, III, IIIA, V, VI, and VIA). Additionally, following the second ATP hydrolysis event, the release of ADP from Pgp is essential to complete the catalytic cycle, i.e. to bring the molecule back to the state where it will bind the next molecule of drug substrate (steps VIB and VII). Finally, this model is consistent with our recent finding that ADP release at steps IV and VII (16) appear to be the rate-limiting steps in the catalytic cycle.



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Fig. 8.   A revised scheme for the catalytic cycle of Pgp. The revised scheme is based on the data presented in this paper as well as previous published work by us and others (8-10, 41). The ellipses represent the substrate-binding sites, the "ON" and the "OFF" site. The hexagon portrays the "ON" site with reduced affinity for the drug. Two circles represent the ATP sites, and the circles are shown overlapping to indicate that both sites are required for ATP hydrolysis. The empty square with rounded edges represents the ATP site with reduced affinity for nucleotide. Step I, substrate binds to the "ON" site of Pgp, and ATP binds to one or both of the two ATP sites. Step II, ATP is hydrolyzed, and the drug is possibly moved to the lower affinity "OFF" site. Step III, Pi is released, and the drug extruded from Pgp at this step. Step IIIA, when Pi is replaced by Vi, the Pgp·ADP·Vi complex is generated, which exhibits a reduced affinity for both substrate as well as nucleotide. Step IV, the ADP and Vi dissociate from the complex. Although the ATP sites have reverted to the "high affinity" state, the affinity for drug substrate continues to be low. Step V, following dissociation of the ADP in step IV, an additional molecule(s) of ATP is hydrolyzed. Step VI, Pi is released. Step VIA, when Pi is replaced by Vi, the Pgp·ADP·Vi complex is generated, which continues to exhibit a reduced affinity for substrate. Steps VIB/VII, the dissociation of ADP allows the conformation of Pgp to be restored to its original state with high affinity for substrate binding. The initiation of the next catalytic cycle follows. Although we depict ATP binding and hydrolysis as occurring in ATP site in the N-half of Pgp, our data do not suggest any preference in the presence of saturating concentrations of ATP or 8-azido-ATP, for either of the two sites, and the process appears to be random (see Fig. 5C). The observed drastically reduced affinity of nucleotides for the second ATP site, while the first one is in the catalytic conformation (steps II and V), provides a basis for the proposed alternate catalytic cycle model (9). In addition, our recent work (16) indicates that ADP release at steps IV and VII (underlined) appears to be the rate-limiting steps in the catalytic cycle of ATP hydrolysis.

The model we propose invokes two hydrolysis events (Fig. 8, steps II and V) during each catalytic cycle. We have also demonstrated here that the two hydrolysis events in a single catalytic cycle are kinetically identical (Figs. 5B and 7) and differ only with respect to the status of the molecule vis à vis substrate binding (Fig. 6). This too is consistent with the notion that ATP does not show preferential affinity to either ATP site, and the sites are recruited randomly for hydrolysis (see above). The recent resolution of the crystal structure of the soluble ATP subunit, ArsA of the bacterial arsenite efflux pump (37) and ATP subunits of bacterial ABC transporters MutS (38, 39) and MalK (40) that exhibit structural and functional similarity to Pgp, shows that the two functional ATP sites, are each composed of residues from both the N- and the C-terminal ATP sites. Such a tertiary structural organization is plausible for Pgp where the nucleotide binding domains in the N- and C-terminal halves of the protein each contribute to both ATP binding and hydrolysis with similar kinetic properties. The resolution of the structure of the Pgp ATP domains and further work on site-directed mutagenesis of residues in ATP sites should provide additional insights into the mechanism of the catalytic cycle of ATP hydrolysis.


    ACKNOWLEDGEMENTS

We thank Dr. Michael M. Gottesman for discussions and encouragement and Dr. Marianna Müller for critical reading of the manuscript.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Cell Biology, Division of Basic Sciences, NCI, Bldg. 37, Rm. 1B-22, National Institutes of Health, 37 Convent Dr., Bethesda, MD 20892-4255. Tel.: 301-402-4178; Fax: 301-435-8188; E-mail: ambudkar@helix.nih.gov.

Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M011294200


    ABBREVIATIONS

The abbreviations used are: Pgp, P-glycoprotein; ABC, ATP-binding cassette; AMPPNP, 5'-adenylylimididiphosphate; [125I]IAAP, [125I]io- doarylazidoprazosin; PAGE, polyacrylamide gel electrophoresis; Vi, vanadate; TNP-ATP, 2'-O-(trinitrophenyl)adenosine 5'-triphosphate; MDR, multidrug resistance; MES, 2-[N-morpholino]ethane sulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Gottesman, M. M., Pastan, I., and Ambudkar, S. V. (1996) Curr. Opin. Genet. & Dev. 6, 610-617[CrossRef][Medline] [Order article via Infotrieve]
2. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427[CrossRef][Medline] [Order article via Infotrieve]
3. Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., and Gottesman, M. M. (1999) Annu. Rev. Pharmacol. Toxicol. 39, 361-398[CrossRef][Medline] [Order article via Infotrieve]
4. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992) J. Biol. Chem. 267, 4854-4858[Abstract/Free Full Text]
5. Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476[Abstract]
6. Urbatsch, I. L., al-Shawi, M. K., and Senior, A. E. (1994) Biochemistry 33, 7069-7076[Medline] [Order article via Infotrieve]
7. Ford, J. M., and Hait, W. N. (1990) Pharmacol. Rev. 42, 155-199[Medline] [Order article via Infotrieve]
8. Dey, S., Ramachandra, M., Pastan, I., Gottesman, M. M., and Ambudkar, S. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10594-10599[Abstract/Free Full Text]
9. Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289[CrossRef][Medline] [Order article via Infotrieve]
10. Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520[Abstract/Free Full Text]
11. Ambudkar, S. V., Cardarelli, C. O., Pashinsky, I., and Stein, W. D. (1997) J. Biol. Chem. 272, 21160-21166[Abstract/Free Full Text]
12. Shapiro, A. B., and Ling, V. (1998) Eur. J. Biochem. 254, 189-193[Abstract]
13. Ramachandra, M., Ambudkar, S. V., Chen, D., Hrycyna, C. A., Dey, S., Gottesman, M. M., and Pastan, I. (1998) Biochemistry 37, 5010-5019[CrossRef][Medline] [Order article via Infotrieve]
14. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve]
15. al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992[Abstract/Free Full Text]
16. Kerr, K. M., Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 8657-8664[Abstract/Free Full Text]
17. Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647[Medline] [Order article via Infotrieve]
18. Lerner-Marmarosh, N., Gimi, K., Urbatsch, I. L., Gros, P., and Senior, A. E. (1999) J. Biol. Chem. 274, 34711-34718[Abstract/Free Full Text]
19. Liu, R., and Sharom, F. J. (1997) Biochemistry 36, 2836-2843[CrossRef][Medline] [Order article via Infotrieve]
20. Hrycyna, C. A., Ramachandra, M., Germann, U. A., Cheng, P. W., Pastan, I., and Gottesman, M. M. (1999) Biochemistry 38, 13887-13899[CrossRef][Medline] [Order article via Infotrieve]
21. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 22957-22961[Abstract/Free Full Text]
22. Muller, M., Bakos, E., Welker, E., Varadi, A., Germann, U. A., Gottesman, M. M., Morse, B. S., Roninson, I. B., and Sarkadi, B. (1996) J. Biol. Chem. 271, 1877-1883[Abstract/Free Full Text]
23. Loo, T. W., and Clarke, D. M. (1995) J. Biol. Chem. 270, 21449-21452[Abstract/Free Full Text]
24. Senior, A. E., and Bhagat, S. (1998) Biochemistry 37, 831-836[CrossRef][Medline] [Order article via Infotrieve]
25. al-Shawi, M. K., and Senior, A. E. (1993) J. Biol. Chem. 268, 4197-4206[Abstract/Free Full Text]
26. Senior, A. E., and Gadsby, D. C. (1997) Semin. Cancer Biol. 8, 143-150[CrossRef][Medline] [Order article via Infotrieve]
27. Senior, A. E. (1998) Acta Physiol. Scand. Suppl. 643, 213-218[CrossRef][Medline] [Order article via Infotrieve]
28. Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) J. Bioenerg. Biomembr. 27, 31-36[Medline] [Order article via Infotrieve]
29. Sharom, F. J., Yu, X., Chu, J. W., and Doige, C. A. (1995) Biochem. J. 308, 381-390[Medline] [Order article via Infotrieve]
30. Scarborough, G. A. (1995) J. Bioenerg. Biomembr. 27, 37-41[Medline] [Order article via Infotrieve]
31. Urbatsch, I. L., Gimi, K., Wilke-Mounts, S., and Senior, A. E. (2000) Biochemistry 39, 11921-11927[CrossRef][Medline] [Order article via Infotrieve]
32. Sankaran, B., Bhagat, S., and Senior, A. E. (1997) Biochemistry 36, 6847-6853[CrossRef][Medline] [Order article via Infotrieve]
33. Urbatsch, I. L., Sankaran, B., Bhagat, S., and Senior, A. E. (1995) J. Biol. Chem. 270, 26956-26961[Abstract/Free Full Text]
34. Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995) J. Biol. Chem. 270, 19383-19390[Abstract/Free Full Text]
35. Urbatsch, I. L., Gimi, K., Wilke-Mounts, S., and Senior, A. E. (2000) J. Biol. Chem. 275, 25031-25038[Abstract/Free Full Text]
36. Liu, R., and Sharom, F. J. (1996) Biochemistry 35, 11865-11873[CrossRef][Medline] [Order article via Infotrieve]
37. Zhou, T., Radaev, S., Rosen, B. P., and Gatti, D. L. (2000) EMBO J. 19, 4838-4845[Abstract/Free Full Text]
38. Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710[CrossRef][Medline] [Order article via Infotrieve]
39. Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717[CrossRef][Medline] [Order article via Infotrieve]
40. Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961[Abstract/Free Full Text]
41. Urbatsch, I. L., Beaudet, L., Carrier, I., and Gros, P. (1998) Biochemistry 37, 4592-4602[CrossRef][Medline] [Order article via Infotrieve]


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