Functionally Similar Vanadate-induced 8-Azidoadenosine 5'-[alpha -32P]Diphosphate-trapped Transition State Intermediates of Human P-glycoprotein Are Generated in the Absence and Presence of ATP Hydrolysis*

Zuben E. Sauna, Melissa M. Smith, Marianna Müller, and Suresh V. AmbudkarDagger

From the Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255

Received for publication, January 30, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha -32P]8-azido-ADP trapping into Pgp in the presence of [alpha -32P]8-azido-ATP (with ATP hydrolysis) or [alpha -32P]8-azido-ADP (without ATP hydrolysis). Vi mimics Pi to trap the nucleotide tenaciously in the Pgp·[alpha -32P]8-azido-ADP·Vi conformation in either condition. Thus, by using [alpha -32P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP. The trapped [alpha -32P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha -32P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([125I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha -32P]8-azido-ADP (in the absence of hydrolysis) displays an ~2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha -32P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp·[alpha -32P]8-azido-ADP (or ADP)·Vi transition state complexes generated either in the absence of or accompanying [alpha -32P]8-azido-ATP hydrolysis are functionally indistinguishable.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cancer cells resistant to chemically diverse drugs with multiple mechanisms of action are defined as exhibiting the multiple drug resistance (MDR)1 phenotype. The best defined form of MDR in human cells is due to the overexpression of P-glycoprotein (Pgp). This 170-kDa plasma membrane protein is a member of the ATP-binding cassette (ABC) superfamily of transport proteins, and can extrude a range of hydrophobic anticancer drugs from cells against a concentration gradient and thus render cells resistant to cytotoxic chemotherapeutic agents. Analysis of hydropathy plots suggests that Pgp consists of two homologous halves each containing six transmembrane helices and one nucleotide-binding or ATP site in each half (1, 2).

The extrusion of cytotoxic agents is powered by ATP hydrolysis, and the ATPase activity of Pgp has been studied in considerable detail. [alpha -32P]8-azido-ATP, a radiolabeled, photoaffinity analogue of ATP, has proved to be a valuable reagent in understanding interactions between nucleotide and Pgp (3-7). The use of [alpha -32P]8-azido-ATP along with orthovanadate (Vi) has permitted experimental strategies to elucidate the catalytic cycle (3, 5, 7-12). Vi inhibits Pgp ATPase activity by trapping nucleotide in the catalytic site to generate the transition state conformation, Pgp·ADP·Vi. It has been established that it is always a nucleoside diphosphate that is the trapped species (3, 13). Thus if ATP (or 8-azido-ATP) is used to initiate the reaction, at least one turnover of ATP hydrolysis, converting ATP to ADP, is essential for trapping to occur. This has allowed Vi-trapping experiments to be used to construct a catalytic scheme for ATP hydrolysis by Pgp.

Our recent work (10-12) has considerably expanded the original model for the catalytic scheme of Pgp proposed by Senior and co-workers (14). Our data suggest that ATP hydrolysis at one of the two ATP sites results in a dramatic conformational change where the affinities of both the substrate and the nucleotide for Pgp are drastically reduced. The fact that ATP binding to the second site is arrested while the first one is in a catalytic conformation appears to be the basis for alternate catalysis in Pgp (12). Moreover, for Pgp to regain the conformation that binds substrate with high affinity, the hydrolysis of an additional molecule of nucleotide is obligatory. Finally, we showed that release of ADP from the Pgp·ADP·Pi transition state is the rate-limiting step in the catalytic cycle of ATP hydrolysis (11).

Much of the work described above has benefited from the availability of [alpha -32P]8-azido-ATP allowing direct visualization and quantification of the nucleotide interaction with Pgp. However, as only the photoaffinity analogue of nucleoside triphosphate was available as a radioisotope, this has precluded directly addressing many questions that require the 32P-labeled azido derivative of nucleoside diphosphate. In this study, we have characterized for the first time the binding and Vi-induced trapping of [alpha -32P]8-azido-ADP to Pgp. We demonstrate that [alpha -32P]8-azido-ADP binds specifically to Pgp with a Kd comparable to that for [alpha -32P]8-azido-ATP. The kinetic scheme for the Vi-induced inhibition of Pgp ATP hydrolysis proposed by Senior's group (14, 15) suggests that incorporation of ADP into the Pgp·ADP·Vi ternary complex may occur either following hydrolysis of ATP to ADP or directly by the addition of ADP in the absence of hydrolysis.

We demonstrate in this study that it is possible to initiate Vi-induced trapping with either [alpha -32P]8-azido-ADP or [alpha -32P]8-azido-ATP, with similar kinetics, and that the trapped [alpha -32P]8-azido-ADP distributed equally between the N- and the C-terminal halves of Pgp under both hydrolysis and non-hydrolysis conditions. Vi-induced trapping under both hydrolysis and non-hydrolysis conditions exhibited the same requirement for divalent cations. Previous work has demonstrated that a point mutation in the Walker B region (D555N and D1200N, respectively) of either the N- or C-terminal ATP sites arrests ATP hydrolysis and Vi-induced trapping (6, 9). We find that these mutants also do not show Vi-induced [alpha -32P]8-azido-ADP trapping into Pgp in the absence of hydrolysis, suggesting that the functional interaction of both ATP sites is necessary for the formation of the transition state intermediate. Our previous work demonstrated a direct interaction between the substrate and ATP sites by showing that the Vi-trapped intermediate exhibits drastically reduced affinity for the Pgp substrate analogue, [125I]iodoarylazidoprazosin (IAAP) (5, 8, 10). We show here that the Vi-trapped intermediate generated with 8-azido-ADP, 8-azido-ATP, ADP, and ATP results in drastically reduced binding of IAAP to Pgp. Most interesting, however, analyses of the Arrhenius plots at steady state demonstrate that the activation energy for generating the Pgp·[alpha -32P]8-azido-ADP·Vi transition state complex starting with [alpha -32P]8-azido-ADP is ~2.5 times greater than if [alpha -32P]8-azido-ATP were used, and it is 1.5 times greater than that required for the basal or verapamil-stimulated hydrolysis of ATP or 8-azido-ATP. These data indicate that the Vi-trapped intermediate generated under hydrolysis or non-hydrolysis conditions do not show functional differences, and we suggest that the use of [alpha -32P]8-azido-ADP should prove useful to compare the catalytic cycle of ATP hydrolysis by different ABC transporters.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- [125I]IAAP, 2,200 Ci/mmol, was obtained from PerkinElmer Life Sciences. [alpha -32P]8-Azido-ATP (15-20 Ci/mmol), [alpha -32P]8-azido-ADP (15-20 Ci/mmol), 8-azido-ATP, and 8-azido-ADP were purchased from Affinity Labeling Technologies, Inc. Pgp-specific monoclonal antibody C219 was a generous gift from Fujirebio Diagnostics Inc. (Malvern, PA). 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 (His6)) as described (5). Crude membranes were prepared as described previously (5, 16).

Preparation of Crude Membranes from HeLa Cells Expressing Mutant and Wild-type Pgp-- A 70-80% confluent monolayer of HeLa cells was infected with vTF7-3 and transfected with pTM1-MDR1 (wild type) or pTM1-MDR1 bearing the homologous mutations at positions 555 (D555N) and 1200 (D1200N) as described previously (9). The vaccinia virus expression vectors pTM1-MDR1 (wild type) and pTM1-MDR1(D555N) and pTM1-MDR1(D1200N) were provided by C. A. Hrycyna and M. M. Gottesman, Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda. Crude membranes were prepared from the HeLa cells as described previously (17).

Purification and Reconstitution of Pgp-- Human Pgp from crude membranes of High Five insect cells was purified as described (5). The crude membranes were solubilized with octyl beta -D-glucopyranoside (1.25%) in the presence of 20% glycerol and a 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 the 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 (5) and quantified by Amido Black protein estimation method as described previously (18). Purified Pgp was reconstituted into proteoliposomes by dialysis as described (11).

Photoaffinity Labeling with IAAP-- The crude membranes (10-50 µg) were incubated at room temperature in 50 mM Tris-HCl, pH 7.5, with IAAP (3-6 nM) for 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 a STORM 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT.

Binding of [alpha -32P]8-Azido-ATP or [alpha -32P]8-Azido-ADP to Pgp-- Crude membranes (1-2 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 10 µM [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP (8-10 µCi/nmol) in the dark on ice for 5 min. The samples were then cross-linked by UV illumination at 365 nm on ice as described above. Ice-cold ATP (12.5 mM) was added to displace excess non-covalently bound radionucleotide. Excess unbound nucleotides were removed by centrifugation at 300,000 × g at 4 °C for 10 min by using a S120-AT2 rotor in a RC-M120EX micro-ultracentrifuge (Sorvall, Newtown, CT), and the pellet was resuspended in SDS-PAGE sample buffer. Following SDS-PAGE on a 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 as described above.

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 (10). Crude membranes (1-2 mg/ml) were incubated in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the dark for 6-10 min as specified in the figure legends at 37 °C. In some experiments instead of [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP, 8-azido-ATP, 8-azido-ADP, ATP, or ADP were used at concentrations indicated in the legend to Fig. 7. The reaction was stopped by adding 12.5 mM ice-cold ATP and placing the sample immediately on ice. The samples were then cross-linked by UV illumination at 365 nm on ice for 10 min.

ATPase Assays-- ATPase activity of Pgp in crude membranes was measured by the end point, Pi assay as described previously (5, 11), with minor modifications. Pgp-specific activity was recorded as the Vi-sensitive ATPase activity. The assay measured the amount of inorganic phosphate released over 20-30 min at different temperatures ranging from 22 to 39 °C in the ATPase assay buffer. The assay was carried out under basal conditions or in the presence of verapamil, 30 µM. The reaction was initiated with 5 mM ATP or 2 mM 8-azido-ATP and quenched with SDS (2.5% final concentration); the amount of Pi released was quantitated using a colorimetric method (5). The assay with 8-azido-ATP was performed under subdued lighting.

Binding of TNP-ATP to Pgp-- Binding of the fluorescent ATP 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 protein) were incubated with TNP-ATP, 100 µM, 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) was 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 emission scans 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

Binding of [alpha -32P]8-Azido-ADP to Pgp-- The radiolabeled, photoaffinity analogue of ATP, [alpha -32P]8-azido-ATP, has shown to be extremely useful in understanding the catalytic cycle of ATP hydrolysis by Pgp (3, 5, 12, 19), the ATP binding and hydrolysis of other ABC transporters (20, 21), and in mapping the active site of myosin (22). However, the unavailability of the radiolabeled nucleoside diphosphate, [alpha -32P]8-azido-ADP, until recently has precluded many direct experiments to address questions about the catalytic cycle of Pgp. In this study, we characterize the kinetics of [alpha -32P]8-azido-ADP binding to Pgp, and we demonstrate its usefulness as a reagent to study the mechanism of action of Pgp. Fig. 1A shows that [alpha -32P]8-azido-ADP binds specifically to the ATP site of Pgp as the binding is completely competed by the nucleotides, ATP and ADP, as well as the nucleotide analogues, 8-azido-ATP and 8-azido-ADP. Additionally, Fig. 1B demonstrates that the binding of [alpha -32P]8-azido-ADP is saturable with a Kd of 10 ± 3 µM, which is very similar to the Kd of [alpha -32P]8-azido-ATP binding to Pgp, which is in the range of 10-15 µM (12). These results suggest that [alpha -32P]8-azido-ADP binds directly to Pgp in a manner similar to [alpha -32P]8-azido-ATP.


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Fig. 1.   [alpha -32P]8-azido-ADP binds specifically to the ATP sites of Pgp. A, binding of [alpha -32P]8-azido-ADP to Pgp is inhibited by nucleotides and nucleotide analogues. 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 10 µM [alpha -32P]8-azido-ADP (8-10 µCi/nmol) in the dark for 5 min at 37 °C. Samples were then either not treated with a nucleotide (control) or were treated with 2 mM ATP, 2 mM ADP, 1 mM 8-azido-ATP, or 1 mM 8-azido-ADP for 5 min at 4 °C in the dark followed by UV irradiation at 365 nm as described under "Experimental Procedures." Samples were centrifuged and resuspended in 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 36 h. Autoradiogram shows control membranes (lane 1) or membranes pretreated with 2 mM ATP (lane 2), 2 mM ADP (lane 3), 1 mM 8-azido-ATP (lane 4), or 1 mM 8-azido-ADP (lane 5). Equal amounts of crude membranes (96 µg of protein) were loaded in each lane. B, [alpha -32P]8-azido-ADP exhibits saturation binding to Pgp. Crude membranes (5 mg/ml) were incubated in the ATPase assay buffer containing increasing concentrations of [alpha -32P]8-azido-ADP (10 µCi/nmol) in the dark for 5 min at 4 °C, cross-linked by UV irradiation, and followed by SDS-PAGE as described under "Experimental Procedures." Following SDS-PAGE, the gel was dried, and the radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager and the software ImageQuaNT. The line represents the best fit for the data by non-linear least squares regression analysis using the software GraphPad Prism 2.0 for the PowerPC Macintosh. The affinity (Kd) of [alpha -32P]8-azido-ADP for Pgp was 10 ± 3 µM.

Vi-induced Trapping of [alpha -32P]8-Azido-ADP into Pgp-- It has been established that the ATPase activity of Pgp follows simple Michaelis-Menten kinetics with a Km of ~0.3 to 1 mM depending on the source of Pgp (11, 23-25). The technical difficulties that this low affinity for nucleotide entails have been enumerated in detail by Senior and colleagues (24, 26), and the Vi-induced trapping of nucleotides into the ATP sites has proved to be an invaluable tool in elucidating the ATP hydrolysis cycle of Pgp. Vi is a potent inhibitor of the ATPase activity of Pgp, which acts by mimicking the pentacovalent phosphorus catalytic transition state, thereby trapping the nucleotide tenaciously. This transition state conformation of Pgp has been characterized in earlier studies (3), by incubation of Pgp with [alpha -32P]8-azido-ATP and Vi at 37 °C (i.e. allowing [alpha -32P]8-azido-ATP to be hydrolyzed to [alpha -32P]8-azido-ADP). Several lines of evidence have indicated that the trapped moiety is the nucleoside diphosphate (3, 4, 13), and Fig. 2A (after Senior et al. (14)) depicts the kinetic scheme for generating the Pgp·MgADP·Vi complex starting with Pgp, MgATP, and Vi. ATP binds to Pgp in the presence of Mg2+ (step 1). and this is followed by hydrolysis (step 2) during which ATP is converted to ADP and Pi. Subsequently Pi is released and ADP dissociates from Pgp. However, if Vi, an analogue of Pi is present, it "traps" the ADP to form a stable, ternary, non-covalent complex, Pgp·MgADP·Vi (step 3). This complex eventually dissociates to Pgp + MgADP + Vi (step 4). As step 4 is a reversible process, in principle if MgADP were directly provided to Pgp, it would be stabilized by Vi into the Pgp·MgADP·Vi transition state complex (step 4b in boxed figure). Fig. 2B shows that if Pgp is incubated with 50 µM [alpha -32P]8-azido-ADP and 250 µM Vi for 10 min at 37 °C, the [alpha -32P]8-azido-ADP is occluded at the catalytic site (lane 3) and is not competed out by even 200-fold excess non-radioactive nucleotide (lane 4). This is in stark contrast to incubating Pgp in the presence of [alpha -32P]8-azido-ADP but in the absence of Vi, where there is significantly less incorporation of [alpha -32P]8-azido-ADP (lane 1) reflecting the binding of nucleotide to Pgp which is competed by excess ATP (lane 2). Note that for the panel showing incorporation of [alpha -32P]8-azido-ADP in the absence of Vi, the gel was exposed to the x-ray film for 4 times as much time as the panel showing incorporation of [alpha -32P]8-azido-ADP in the presence of Vi. The autoradiogram also demonstrates that while the [alpha -32P]8-azido-ADP incorporated in the absence of Vi can be competed out by excess ATP (lane 2), the [alpha -32P]8-azido-ADP incorporated in the presence of Vi is unaffected by excess ATP (lane 4). Thus, these experiments show that it is possible to trap directly [alpha -32P]8-azido-ADP into Pgp suggesting that hydrolysis is not a prerequisite for Vi-induced trapping per se but is obligatory when [alpha -32P]8-azido-ATP (or any other hydrolyzable nucleoside triphosphate) is used so as to generate the nucleoside diphosphate.


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Fig. 2.   [alpha -32P]8-Azido-ADP in the presence of Vi is trapped into a ternary complex with Pgp. A, catalytic scheme for the Vi-induced trapping of ADP into Pgp. The kinetic scheme presented here is based on published reports (11, 14, 27) Step 1, MgATP binds to Pgp in the presence of Mg2+. Step 2, binding of MgATP is followed by hydrolysis; MgATP is converted to MgADP; Pi is released, and MgADP dissociates from Pgp. Step 3, however, if Vi, an analogue of Pi, is present in the reaction, MgADP is trapped to form a stable, ternary, non-covalent complex, Pgp·MgADP·Vi. Step 4, eventually, MgADP and Pi dissociate from Pgp. As Step 4 is a reversible process, in principle if MgADP were directly provided to Pgp then the Pgp·MgADP formed would be stabilized by Vi into the Pgp·MgADP·Vi transition state complex. This is depicted in the box at the bottom (Steps 4a and 4b) except that MgADP is replaced with Mg[alpha -32P]8-azido-ADP (see B). Note: although we depict the release of Pi from the complex as concurrent with ATP hydrolysis (Step 2), it is very likely that there are several sub-states between the hydrolysis of MgATP and the release of Pi. More importantly, each of these could have subtle conformational differences, and agents such as Vi, aluminum fluoride, or beryllium fluoride could trap each of these in a ternary complex. B, [alpha -32P]8-azido-ADP incubated with Pgp in the presence of Vi results in [alpha -32P]8-azido-ADP being trapped into a ternary complex. Crude membranes (2 mg/ml) were incubated at 37 °C in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ADP (2-4 µCi/nmol) either in the absence or presence of 250 µM Vi in the dark for 10 min. The reaction was stopped either by the addition of 10 mM ice-cold ATP and placing the sample on ice or by placing the sample on ice without adding ATP. The samples were then cross-linked by UV irradiation at 365 nm, and following SDS-PAGE on an 8% Tris glycine gel at constant voltage, the gels were dried and exposed to Bio-Max MR film at -70 °C. The autoradiogram shows the following: lane 1, membranes treated with 50 µM [alpha -32P]8-azido-ADP; lane 2, membranes treated with 50 µM [alpha -32P]8-azido-ADP and 12.5 mM ATP was added prior to UV cross-linking; lane 3, membranes treated with 50 µM [alpha -32P]8-azido-ADP and 250 µM Vi; lane 4, membranes treated with 50 µM [alpha -32P]8-azido-ADP and 250 µM Vi, and 12.5 mM ATP was added prior to UV cross-linking. The left and right panels are autoradiograms from the same gel; however, as the signals for lanes 3 and 4 were extremely high the gels were exposed to the x-ray film for different times. The left panel (lanes 1 and 2) were exposed to the x-ray film for 36 h at -70 °C and the right panel (lanes 3 and 4) for 8 h. An equal amount of protein (96 µg) was loaded in each lane. C, distribution of trapped [alpha -32P]8-azido-ADP in the N- and the C-terminal ATP sites of Pgp. Crude membranes (2 mg/ml) were incubated in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the dark for 10 min at 37 °C and cross-linked by UV irradiation (365 nm). In a parallel experiment, the crude membranes were incubated in an identical manner with 50 µM [alpha -32P]8-azido-ATP (2-4 µCi/nmol) and 250 µM Vi. Samples were then either treated with trypsin (protein/trypsin, 1:10) for 5 min at 37 °C to separate the N- and C-terminal halves of Pgp as described previously (8) or incubated at 37 °C in the absence of trypsin. To both untreated and trypsin-treated samples, SDS-PAGE sample buffer containing 5 M urea was added. 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 16-24 h. Autoradiogram shows the following: lane 1, control membranes incubated with [alpha -32P]8-azido-ATP and Vi; lane 2, membranes incubated with [alpha -32P]8-azido-ATP and Vi and treated with trypsin; lane 3, control membranes incubated with [alpha -32P]8-azido-ADP and Vi; and lane 4, membranes incubated with [alpha -32P]8-azido-ADP and Vi and treated with trypsin. An equal quantity of crude membranes (58 µg protein) was loaded in each lane. The N- and C-terminal halves of Pgp were identified by immunoblotting with antibodies specific to the N- and C-terminal regions of Pgp (data not shown) as described previously (7, 8).

To characterize further Pgp in the transition state conformation generated by using [alpha -32P]8-azido-ADP, we compared the distribution of the trapped [alpha -32P]8-azido-ADP in the N- and the C-terminal ATP sites of Pgp. Fig. 2C demonstrates that consistent with previously published reports (9, 12, 27-29), the [alpha -32P]8-azido-ADP distributes approximately equally between the N- and C-terminal halves of Pgp, and the distribution is similar regardless of whether the occluded [alpha -32P]8-azido-ADP is generated through the hydrolysis of [alpha -32P]8-azido-ATP or directly providing the nucleoside diphosphate itself.

Effect of Divalent Cations on the Vi-induced Trapping of [alpha -32P]8-Azido-ADP Under Hydrolysis and Non-hydrolysis Conditions-- The experiments described thus far have used magnesium as a metal cofactor with the nucleotide as it is well established that ATP binds to Pgp as an MgATP complex (30). However, several divalent cations such as manganese and cobalt are known to support ATPase activity (31), although with considerably reduced Vmax values vis-à-vis magnesium. The Vmax value for MnATPase is 43% that for MgATPase and only 10% for CoATPase (3). Studies have also demonstrated that replacing magnesium with other cations such as Mn2+ and Co2+ also support the Vi-induced trapping of [alpha -32P]8-azido-ADP (11, 27, 31). Compared with the extent of Vi-induced [alpha -32P]8-azido-ADP trapping in the presence of Mg2+, Mn2+, and Co2+, the amount of [alpha -32P]8-azido-ADP incorporated in the presence of Ca2+ was negligible (31). In Fig. 3 we compared the Vi-induced trapping of [alpha -32P]8-azido-ADP under hydrolysis conditions (i.e. initiating the reaction with [alpha -32P]8-azido-ATP and Vi) and non-hydrolysis conditions (i.e. initiating the reaction with [alpha -32P]8-azido-ADP and Vi). We find that Mg2+, Mn2+, and Co2+ permit Vi-induced trapping of [alpha -32P]8-azido-ADP under both hydrolysis and non-hydrolysis conditions. The extent of trapping under both conditions follows the pattern Mn2+ > Co2+ > Mg2+. Ca2+ does not support Vi-induced trapping of [alpha -32P]8-azido-ADP even in the absence of hydrolysis.


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Fig. 3.   Effect of divalent cations on the incorporation of [alpha -32P]8-azido-ADP into Pgp during Vi-induced trapping. Crude membranes were incubated with 50 µM [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the presence of indicated cations (5 mM) for 10 min at 37 °C in the dark. The reaction was stopped by adding 200-fold excess ATP and transferring to ice followed by cross-linking by UV irradiation. Following SDS-PAGE (58 µg of protein was loaded in each lane), the gels were dried, and the radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager. The filled bars represent [alpha -32P]8-azido-ADP incorporated when trapping is carried out under hydrolysis conditions by incubation with [alpha -32P]8-azido-ATP and Vi and the empty bars represent nucleotide trapping carried out under non-hydrolysis conditions by incubation with [alpha -32P]8-azido-ADP and Vi.

Comparing the Kinetics of Generating the Pgp·[alpha -32P]8-Azido-ADP·Vi Transition State Intermediates Using Either [alpha -32P]8-Azido-ATP or [alpha -32P]8-Azido-ADP and Vi-- We have shown above that it is possible to generate the Pgp·[alpha -32P]8-azido-ADP·Vi transition state intermediate by directly incubating Pgp with [alpha -32P]8-azido-ADP and Vi. It would be important to determine the kinetics of Vi-induced [alpha -32P]8-azido-ADP trapping under hydrolysis and non-hydrolysis conditions. The kinetics of trapping using [alpha -32P]8-azido-ATP + Vi or [alpha -32P]8-azido-ADP + Vi are depicted in Fig. 4, A and B. The incorporation of [alpha -32P]8-azido-ADP through the hydrolysis of increasing concentrations of [alpha -32P]8-azido-ATP or in the presence of increasing concentrations of [alpha -32P]8-azido-ADP itself exhibits Michaelis-Menten kinetics with a Km of 20 ± 4.3 µM for [alpha -32P]8-azido-ATP and a Kd of 16 ± 1.6 µM for [alpha -32P]8-azido-ADP, respectively. Thus, the affinity of [alpha -32P]8-azido-ADP for Pgp is similar under hydrolysis and non-hydrolysis conditions.


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Fig. 4.   Kinetics of Vi-induced trapping of [alpha -32P]8-azido-ADP into Pgp under hydrolysis and non-hydrolysis conditions. A, kinetics of [alpha -32P]8-azido-ADP trapping into Pgp under hydrolysis conditions by using [alpha -32P]8-azido-ATP and Vi. Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer containing increasing concentrations of [alpha -32P]8-azido-ATP (2-4 µCi/nmol) and 250 µM Vi in the dark for 10 min at 37 °C. The reaction was stopped by the addition of 12.5 mM ice-cold ATP and placing the sample on ice. The samples were subjected to UV cross-linking as described above. Following SDS-PAGE, the gel was dried, and the radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager. The line represents a fit of the Michaelis-Menten model to the data by non-linear least squares regression analysis using the software GraphPad Prism 2.0 for the PowerPC Macintosh. The Km of [alpha -32P]8-azido-ATP for Pgp in the presence of Vi was 20 ± 4.3 µM. B, kinetics of [alpha -32P]8-azido-ADP trapping into Pgp using [alpha -32P]8-azido-ADP and Vi. Crude membranes (2 mg/ml) were incubated in the ATPase assay buffer containing increasing concentrations of [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the dark for 10 min at 37 °C. The reaction was stopped by the addition of 10 mM ice-cold ATP and placing the sample immediately on ice. Samples were cross-linked by UV irradiation at 365 nm and processed as described in A. The apparent affinity (Kd) of [alpha -32P]8-azido-ADP for Pgp in the presence of Vi was 16 ± 1.6 µM.

The Vi-induced ADP-trapped Conformation of Pgp Exhibits a Marked Decrease in Affinity for the Fluorescent Nucleotide TNP-ATP-- We have demonstrated earlier that there is a reduced binding of nucleotide to Pgp in the transition state conformation (12). This can be demonstrated by first generating the Pgp·ADP·Vi transition state by incubating with ATP and Vi, washing off excess ATP and Vi, and then determining the extent of binding of TNP-ATP, a hydrolyzable, fluorescent analogue of ATP (32), previously used to characterize the ATP sites of Pgp (33, 34). Fig. 5 shows that when Pgp is pretreated with Vi and ATP or ADP at 37 °C, to generate the Pgp·ADP·Vi complex, there is a marked decrease in the binding of TNP-ATP to Pgp as evidenced by decreased levels of fluorescence. These results are consistent with our earlier finding that binding of [alpha -32P]8-azido-ATP or TNP-ATP to Pgp is drastically reduced when the transporter is trapped in the transition state conformation (12).


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Fig. 5.   TNP-ATP binding to Pgp in transition state conformation generated by pretreatment with either ADP or ATP and Vi. Purified Pgp was reconstituted into proteoliposomes as described under "Experimental Procedures." Proteoliposomes (25 µg of protein) were incubated with 100 µM TNP-ATP at room temperature for 10 min in the dark, 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 peak fluorescence (530 nm) for TNP-ATP + Pgp, control (filled bars) and TNP-ATP + Pgp pre-trapped with ADP or ATP and Vi (empty bars). The bars represent the mean value of four experiments, and the error bars represent the standard deviation. Pgp was trapped by incubating with either ADP (1.25 mM) or 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 the excess nucleotides and Vi were removed by centrifugation.

Mutations in the Walker B Domain of Either the N- or C-terminal ATP Sites of Pgp Arrest Vi-induced Trapping of [alpha -32P]8-Azido-ADP Either in the Presence or Absence of ATP Hydrolysis-- The experiments described above demonstrate that it is possible to generate the Pgp·[alpha -32P]8-azido-ADP·Vi catalytic state intermediate by directly incubating Pgp with [alpha -32P]8-azido-ADP and Vi, and the kinetics are comparable regardless of whether [alpha -32P]8-azido-ADP or [alpha -32P]8-azido-ATP is used to initiate the Vi-induced trapping. It has also been demonstrated previously that if mutations are made in the conserved Walker B consensus motif in either the N- or C-terminal ATP sites of Pgp at positions Asp-555 and Asp-1200, respectively, which represent the putative magnesium-binding site, these mutants are unable to support ATP hydrolysis as well as the Vi-induced trapping of [alpha -32P]8-azido-ADP generated through the hydrolysis of [alpha -32P]8-azido-ATP (6, 9). If only ATP hydrolysis is affected in the mutants, using [alpha -32P]8-azido-ADP instead of [alpha -32P]8-azido-ATP should allow trapping of the nucleoside diphosphate. To test this hypothesis, we incubated crude membranes containing both wild-type and mutant Pgp with either [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP in the presence of Vi at 37 °C for 10 min. Excess ATP (200-fold) was added to all samples and then cross-linked by UV irradiation. The samples were immunoprecipitated, electrophoresed, and exposed to an x-ray film. Fig. 6 demonstrates that the wild-type Pgp shows comparable Vi-induced trapping of [alpha -32P]8-azido-ADP regardless of whether it is generated through hydrolysis of [alpha -32P]8-azido-ATP or when [alpha -32P]8-azido-ADP is provided to Pgp. Additionally, neither mutant supports Vi-induced trapping of [alpha -32P]8-azido-ADP with either [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP, indicating that both the ATP sites are required for the formation of the transition state conformation of Pgp even in the absence of hydrolysis of 8-azido-ATP or ATP.


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Fig. 6.   The Pgp mutants D555N and D1200N in Walker B domain of ATP sites do not show Vi-induced trapping of [alpha -32P]8-azido-ADP using either [alpha -32P]8-azido-ADP or [alpha -32P]8-azido-ATP. Crude membranes were prepared from HeLa cells transiently expressing the pTM1-MDR1 (designated wild type) and pTM1-MDR1 bearing the homologous mutations in Walker B region of ATP sites at positions 555 and 1200 (designated D555N and D1200N), respectively. It was determined by Western blotting using the monoclonal antibody C219 that wild-type and mutant Pgps were expressed at equivalent levels (data not shown). These crude membranes (60 µg of protein) were incubated in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the dark for 10 min at 37 °C. The reaction was stopped by the addition of ice-cold ATP (10 mM), and the samples were cross-linked by UV irradiation and immunoprecipitated using the human Pgp-specific polyclonal antibody PEPG-13 as described previously (7, 9). Following SDS-PAGE on a 8% Tris glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film at -70 °C for 16-24 h. Autoradiogram shows Vi-induced trapping of [alpha -32P]8-azido-ADP under hydrolysis (lanes 1-3) and non-hydrolysis conditions (lanes 4-6). Lane 1, wild-type Pgp membranes labeled with [alpha -32P]8-azido-ATP; lane 2, D555N membranes labeled with [alpha -32P]8-azido-ATP; lane 3, D1200N membranes labeled with [alpha -32P]8-azido-ATP; lane 4, wild-type membranes labeled with [alpha -32P]8-azido-ADP; lane 5, D555N membranes labeled with [alpha -32P]8-azido-ADP; and lane 6, D1200N membranes labeled with [alpha -32P]8-azido-ADP. An equal amount of crude membranes (60 µg of protein) was immunoprecipitated and loaded into each lane.

The Kinetics of Inhibition of IAAP Labeling to Pgp during Vi-induced Trapping of Nucleotides-- We have reported that by using both ATP and 8-azido-ATP that the Vi-trapped Pgp shows a greatly reduced affinity for the substrate analogue IAAP (5, 8, 10, 12). To demonstrate that trapping the Pgp molecule in the transition state is sufficient to reduce the affinity of substrate analogue IAAP for the transporter and that this relationship is not specific to a particular nucleotide, we used both nucleoside tri- and diphosphates to trap Pgp in the transition state conformation. Crude membranes containing Pgp were incubated either with ATP, ADP, 8-azido-ATP, or 8-azido-ADP (1.25 mM) and 250 µM Vi at 37 °C in the dark. Aliquots were removed at intervals, incubated with IAAP, and cross-linked by UV irradiation. Following SDS-PAGE, the IAAP incorporated into the Pgp bands was quantified using a PhosphorImager. The results, depicted in Fig. 7, A-D, show that trapping of all the nucleotides tested inhibit IAAP binding in the presence of Vi. We have shown earlier that nucleotides in the absence of Vi or Vi in the absence of nucleotide do not affect IAAP binding (10). These results demonstrate the fact that in the presence of Vi and Mg2+, it is the nucleoside diphosphate that is trapped at the ATP site, and the resulting conformational changes are sufficient to effect changes in the substrate-binding site, resulting in the decreased affinity for substrate. Moreover, these data indicate that the transition state conformation of Pgp generated either in the presence or absence of hydrolysis of nucleotide is similar with respect to its effect on the substrate-binding site(s).


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Fig. 7.   Decrease in the binding of substrate (IAAP) to Pgp in the transition state conformation induced by Vi occurs independent of the nucleotide used. Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer containing increasing concentrations of 8-azido-ADP, 8-azido-ATP, ADP or ATP, and 250 µM Vi in the dark for 10 min at 37 °C. The reaction was stopped by the addition of 10 mM ice-cold ATP and placing the sample immediately on ice. IAAP (5 nM) was added to each sample and incubated for 5 min under subdued light. The samples were then cross-linked by UV irradiation (365 nm) for 10 min at room temperature (21-23 °C). Following SDS-PAGE on an 8% Tris glycine gel at constant voltage, gels were dried, and the radioactivity incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager. The following nucleotides were used to initiate the Vi-induced trapping: A, 8-azido-ADP; B, 8-azido-ATP; C, ADP; and D, ATP. The lines depict the best fit for the data by non-linear least squares regression analysis using the software GraphPad Prism 2.0 for the PowerPC Macintosh.

The Inhibition of IAAP Labeling of Pgp during Vi-induced Trapping of 8-Azido-ADP and the Extent of Vi-induced Trapping of [alpha -32P]8-Azido-ADP Are Correlated-- To understand whether there is a cause-effect relationship between Vi-induced trapping of nucleoside diphosphate per se and the inhibition of substrate binding to Pgp, we performed the following two experiments in parallel. Crude membranes were incubated with 1.25 mM 8-azido-ADP and 250 µM Vi. Aliquots were removed at different time intervals, treated with IAAP for 5 min, and cross-linked by UV irradiation. In a parallel experiment the crude membranes were incubated with 50 µM [alpha -32P]8-azido-ADP and 250 µM Vi. Aliquots were removed at different time intervals, and 200-fold excess ATP was added followed by cross-linking with UV irradiation. The results of this experiment depicted in Fig. 8 show that the increased [alpha -32P]8-azido-ADP trapping over time is accompanied by decrease in IAAP binding. The inset shows that the two are inversely correlated (r = 0.92) suggesting that Vi-induced trapping of [alpha -32P]8-azido-ADP at the ATP site induces conformational changes that reduce the affinity of IAAP for Pgp.


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Fig. 8.   Incorporation of [alpha -32P]8-azido-ADP into Pgp during Vi-induced trapping is accompanied by a decrease in the binding of IAAP. Crude membranes (1 mg/ml) were incubated with 1.25 mM 8-azido-ADP and 250 µM Vi in the ATPase assay buffer at 37 °C. Aliquots were removed at time intervals indicated on the x axis, treated with IAAP (5 nM) for 5 min, and cross-linked by UV irradiation. In a parallel experiment, the crude membranes were incubated with 50 µM [alpha -32P]8-azido-ADP, (2-4 µCi/nmol) and 250 µM Vi at 37 °C. Aliquots were removed at the same time intervals as indicated above, and 200-fold excess ATP was added followed by cross-linking by UV irradiation. Following SDS-PAGE, the gels were dried, and the radioactivity incorporated into the Pgp band was quantified as described under "Experimental Procedures." The lines represent the best fit for the data by non-linear least squares regression analysis using the software GraphPad Prism 2.0 for the PowerPC Macintosh and depict binding of IAAP () to Pgp and incorporation of [alpha -32P]8-azido-ADP (black-triangle) into Pgp. Inset compares the extent of [alpha -32P]8-azido-ADP and IAAP incorporated into Pgp at each time point; the two are inversely correlated (r = 0.92).

Determination of the Activation Energies for Vi-induced Trapping Using [alpha -32P]8-Azido-ATP and [alpha -32P]8-Azido-ADP-- The results thus far clearly show the following: (a) that nucleoside diphosphates in the presence of Mg2+ and Vi form a stable, ternary, non-covalent complex at the nucleotide-binding site of Pgp. The nucleoside diphosphate can be provided directly or as a nucleoside triphosphate that can be hydrolyzed in situ to a nucleoside diphosphate. (b) When the nucleoside diphosphate is trapped at the ATP site, the ternary complex manifests a profound conformational change at the substrate-binding site. This suggests that although the nucleotide- and substrate-binding sites are independent, long range interactions acting via conformational changes result in these two sites being functionally coupled. (c) The functional effect on the substrate-binding site is the same regardless of whether a nucleoside di- or triphosphate is used to initiate the trapping of Pgp into the Pgp·ADP·Vi transition state intermediate. This raises the question as to whether the two routes for generating the Pgp·ADP·Vi transition state are thermodynamically comparable. Fig. 9A depicts the effect of temperature on the Vi-induced trapping of [alpha -32P]8-azido-ADP. There is a low level of [alpha -32P]8-azido-ADP trapping at 23 °C, which is significantly increased at 37 °C, when Pgp is incubated with [alpha -32P]8-azido-ATP and Vi for 6 min. However, when [alpha -32P]8-azido-ADP and Vi are used to initiate trapping, there is no detectable trapping at 23 °C but a significantly increased level of incorporation of [alpha -32P]8-azido-ADP is observed at 37 °C.


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Fig. 9.   Determination of the activation energy for Vi-induced trapping of [alpha -32P]8-azido-ADP initiated with [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP. A, Vi-induced trapping of [alpha -32P]8-azido-ADP into Pgp under hydrolysis and non-hydrolysis conditions at room temperature (23 °C) and 37 °C. Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ATP (2-4 µCi/nmol) or [alpha -32P]8-azido-ADP (4 µCi/nmol) in the absence or presence of 250 µM Vi in the dark for 6 min at room temperature (23 °C) or at 37 °C. The reaction was stopped by the addition of 12.5 mM ice-cold ATP and placing the sample immediately on ice followed by cross-linking by UV irradiation (365 nm). Following SDS-PAGE, the gels were dried and exposed to Bio-Max MR film at -70 °C for 12 h. The same amount of protein (63 µg) was loaded into each lane. The reaction conditions are depicted at the top of the autoradiogram. B, comparison of Arrhenius plots of [alpha -32P]8-azido-ADP trapping into Pgp under hydrolysis and non-hydrolysis conditions. Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer containing 50 µM [alpha -32P]8-azido-ATP (4 µCi/nmol) or [alpha -32P]8-azido-ADP (2-4 µCi/nmol) and 250 µM Vi in the dark for 6 min at different temperatures ranging from 22 to 39 °C. The reaction was stopped by the addition of 12.5 mM ice-cold ATP and placing the sample immediately on ice and followed by cross-linking by UV irradiation (365 nm) for 10 min on ice. Following SDS-PAGE, the gels were dried, and the radioactivity incorporated into the Pgp band was quantified as described under "Experimental Procedures." A plot of 1/temperature (K) versus log (extent of [alpha -32P]8-azido-ADP incorporated) was used to calculate the activation energy as described in the legend to Table I for Vi-induced [alpha -32P]8-azido-ADP trapping into Pgp initiated by using [alpha -32P]8-azido-ATP (open circle ) or [alpha -32P]8-azido-ADP (black-triangle).

To understand this difference, we determined the activation energies for these two processes. Fig. 9B depicts Arrhenius plots for Vi-induced trapping using either [alpha -32P]8-azido-ADP or [alpha -32P]8-azido-ATP in the temperature range 22-39 °C. In this temperature range both the plots show no discontinuity, but the slopes are significantly different, and the steeper slope for the formation of the transition state intermediate with [alpha -32P]8-azido-ADP translates to a 2.5-fold higher activation energy (152 kJ/mol). A published report of the activation energy for ATP hydrolysis in crude membranes derived from the Chinese hamster ovary cell line, CHRB30, shows a discontinuity at 21 °C (35). We could not perform our experiments at lower temperatures (<22 °C) as the trapping with [alpha -32P]8-azido-ADP and Vi even at 23 °C was undetectable (cf. Fig. 9A). Thus, if [alpha -32P]8-azido-ADP is trapped into Pgp by incubating [alpha -32P]8-azido-ATP and Vi, allowing the [alpha -32P]8-azido-ATP to be hydrolyzed to [alpha -32P]8-azido-ADP, which is then trapped, the energy barrier is significantly lower than if [alpha -32P]8-azido-ADP is provided directly in the presence of Vi. The possible explanations for this apparently paradoxical result are considered under "Discussion." In addition, Table I lists the activation energies for the basal and substrate (verapamil)-stimulated hydrolysis of ATP and 8-azido-ATP by Pgp, the Vi-induced trapping of [alpha -32P]8-azido-ADP under hydrolysis and non-hydrolysis conditions, and for the binding of [alpha -32P]8-azido-ADP and IAAP to Pgp. The activation energies for the hydrolysis of ATP both in the absence (basal) and in the presence of verapamil are comparable (115.5 and 110.4 kJ/mol) and almost identical to that for the hydrolysis of 8-azido-ATP (100.1 kJ/mol). In addition, the activation energy for ATP hydrolysis by Pgp in crude membranes derived from the Chinese hamster ovary cell line, CHRB30 (98.1 kJ/mol) (35), is comparable to that obtained with human Pgp in this study. These data indicate that in terms of the thermodynamics of the system, ATP hydrolysis by Pgp is independent of the species origin of the protein or the nucleotide (ATP or 8-azido-ATP) and that the drug substrate does not significantly affect the activation energy for nucleotide hydrolysis. Even more intriguing is the fact that the activation energy for the Vi-induced trapping of [alpha -32P]8-azido-ADP under hydrolysis conditions is 62 kJ/mol or approximately half that for the substrate-stimulated ATP hydrolysis. The Vi-induced trapping of [alpha -32P]8-azido-ADP arrests the catalytic cycle after only one hydrolysis event and thus would have an activation energy one-half of that for the entire catalytic cycle. It is also clear from the data in Table I that the activation energies for binding of nucleoside diphosphate or substrate (IAAP) are significantly lower when compared with trapping of nucleotides. Also, whereas either [alpha -32P]8-azido-ADP or [alpha -32P]8-azido-ATP can be used to initiate Vi-induced trapping with very similar kinetics and functional effects, the energy barriers that these two pathways entail are significantly different (152 versus 62 kJ/mol).

                              
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Table I
Activation energies (Eact) for basal and verapamil-stimulated ATP hydrolysis, 8-azido-ATP hydrolysis, Vi-induced [alpha -32P]8-azido-ADP trapping, [alpha -32P]8-azido-ADP binding, and IAAP binding


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Due to the importance of Pgp in cancer chemotherapy and as a model system for ABC transporters in general, the catalytic cycle of this transporter has been studied in considerable detail (for reviews see Refs. 2, 14, and 30). Building upon the model proposed by Senior's group (14), we have recently elucidated the catalytic cycle of Pgp in considerable detail (10-12). The essential features of the cycle are as follows. (i) ATP hydrolysis results in a dramatic conformational change where the affinity of both the substrate and the nucleotide for Pgp is reduced >30-fold. (ii) 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. (iii) The release of ADP from the Pgp·ADP·Pi transition state is the rate-limiting step in the catalytic cycle. These studies have relied heavily on the use of the radiolabeled photoaffinity analogue of ATP, [alpha -32P]8-azido-ATP. The Pgp·[alpha -32P]8-azido-ADP·Vi transition state intermediate has proved to be particularly useful in understanding the catalytic cycle. As illustrated in Fig. 2A, the forward reaction involves the hydrolysis of ATP and the dissociation of Pi. Vi, an analogue of Pi, then traps the ADP into a stable ternary complex, Pgp·ADP·Vi. There is published evidence that the trapped moiety is always the nucleoside diphosphate (3, 13), which is confirmed by directly providing the nucleoside diphosphate in this work. In addition, we have characterized the trapping of the ADP analogue, [alpha -32P]8-azido-ADP, under both hydrolysis and non-hydrolysis conditions.

The results given in Fig. 1, A and B, indicate that [alpha -32P]8-azido-ADP binds specifically to Pgp at the ATP site, similar to ADP. The question as to whether Vi-induced trapping can be effected in the absence of hydrolysis by directly providing the nucleoside diphosphate is addressed in Fig. 2B. We demonstrate that [alpha -32P]8-azido-ADP in the presence of Vi is trapped into the Pgp·[alpha -32P]8-azido-ADP·Vi transition state. This experiment clearly distinguishes [alpha -32P]8-azido-ADP that is trapped in the ternary, Pgp·[alpha -32P]8-azido-ADP·Vi, complex from binding of nucleotide per se. It is clear that in the absence of Vi, [alpha -32P]8-azido-ADP binds to Pgp, which is completely competed by the addition of excess non-radioactive ATP. On the other hand, in the presence of Vi, [alpha -32P]8-azido-ADP is tenaciously trapped in the Pgp·[alpha -32P]8-azido-ADP·Vi complex and under this condition even a 200-fold excess of ATP (Fig. 2B) cannot compete out the nucleoside diphosphate. Previous reports show that, at saturating concentrations of [alpha -32P]8-azido-ATP in the presence of Vi, [alpha -32P]8-azido-ADP was trapped into the N- and C-terminal halves of Pgp (9, 12, 27-29). Fig. 2C demonstrates that there is an almost equal distribution of [alpha -32P]8-azido-ADP into the N- and C-terminal halves regardless of whether the [alpha -32P]8-azido-ADP is trapped under hydrolysis or non-hydrolysis conditions. It has been established previously that Mg2+ or another divalent cation such as Mn2+ or Co2+ are necessary for ATP hydrolysis (31) and Vi-induced trapping of [alpha -32P]8-azido-ADP (27, 31). Consistent with these findings, the data in Fig. 3 demonstrate that Mg2+, Mn2+, and Co2+ permit Vi-induced trapping of [alpha -32P]8-azido-ADP under both hydrolysis and non-hydrolysis conditions.

It is important to determine whether the kinetics of Vi-induced trapping differs under hydrolysis and non-hydrolysis conditions. We show that during the trapping of [alpha -32P]8-azido-ADP via the hydrolysis of [alpha -32P]8-azido-ATP into Pgp, the Km for [alpha -32P]8-azido-ATP is nearly identical to the Kd of [alpha -32P]8-azido-ADP trapping when the nucleoside diphosphate is provided directly to Pgp in the presence of Vi (Fig. 4, A and B). These results provide evidence that [alpha -32P]8-azido-ADP is trapped into Pgp in the presence of Vi in a similar manner under both hydrolysis and non-hydrolysis conditions. In addition, [alpha -32P]8-azido-ADP can be used to delineate the relationship between the two ATP sites in hydrolysis and Vi-induced trapping. Earlier work (6, 9) has shown that mutations in the conserved Walker B consensus motif in either the N- or the C-terminal ATP sites of Pgp arrest ATP hydrolysis as well as the Vi-induced trapping of [alpha -32P]8-azido-ADP generated through the hydrolysis of [alpha -32P]8-azido-ATP. The mutations in either the N-terminal ATP site (D555N) or the C-terminal site (D1200N) abolish Vi-induced trapping of [alpha -32P]8-azido-ADP by both the hydrolysis and non-hydrolysis routes (Fig. 6). These findings demonstrate that both ATP sites are required not only for ATP hydrolysis but also for Vi-induced nucleoside diphosphate trapping even under non-hydrolysis conditions. Thus, except for binding of nucleotide, each ATP site does not appear to be able to carry out subsequent steps in the catalytic cycle without the participation of the other site.

A key aspect of the proposed catalytic cycle of Pgp has been the fact that ATP hydrolysis results in a dramatic conformational change, which results in a drastic decrease in the affinity of both the substrate (5, 8, 10) and the nucleotide (12) for the Vi-trapped intermediate of Pgp. These parameters thus would be important determinants in characterization of the Vi-trapped intermediate generated in the absence of ATP hydrolysis. The Vi-trapped intermediates of Pgp formed both in the absence of and following ATP hydrolysis show reduced affinity for nucleotide, as measured by the binding of the fluorescent analogue, TNP-ATP (Fig. 5). The second characteristic of the Vi-trapped intermediate, viz. that it exhibits a significant decrease in affinity for the drug substrate IAAP, is a direct and quantitative measure of the long range conformational coupling between the drug- and nucleotide-binding sites. Moreover, from the perspective of this study, the long range effect on the substrate-binding site would be a more stringent evaluation of the Vi-trapped conformations obtained via these two routes. We therefore evaluated this aspect in some detail. Thus, trapping Pgp in the transition state conformation with any of these nucleoside di- or triphosphates such as 8-azido-ADP, 8-azido-ATP, ADP, and ATP inhibits IAAP binding to Pgp (see Fig. 7). The 8-azido-ADP and 8-azido-ATP have lower Ki values than ADP and ATP, respectively. Finally, we made simultaneous measurements of [alpha -32P]8-azido-ADP trapping and inhibition of IAAP binding during Vi-induced trapping of [alpha -32P]8-azido-ADP under non-hydrolysis conditions over time. The results show that these two events are inversely correlated, suggesting a cause-effect relationship (Fig. 8). A similar relationship exists when these measurements are made under ATP hydrolysis conditions (data not given).

Since the Vi-trapped intermediates generated under hydrolysis or non-hydrolysis conditions do not show functional differences, we compared the thermodynamics of these two pathways. The data in Fig. 9A demonstrate that under hydrolysis conditions there is significantly greater Vi-induced [alpha -32P]8-azido-ADP trapping at 37 °C than at 23 °C. Under non-hydrolysis conditions, the difference is even more marked as there is no detectable Vi-induced [alpha -32P]8-azido-ADP trapping at 23 °C. Arrhenius plots of the Vi-induced trapping initiated by either [alpha -32P]8-azido-ATP or [alpha -32P]8-azido-ADP show that these pathways have different activation energies (Fig. 9B). When Vi-induced trapping is effected by incubating [alpha -32P]8-azido-ATP in the presence of Vi under ATP hydrolysis conditions, the activation energy is 62 kJ/mol (mean value of three experiments). Conversely, when [alpha -32P]8-azido-ADP is directly trapped in the presence of Vi without ATP hydrolysis, the activation energy is 152 kJ/mol (mean value of three experiments). Thus, the latter route of generating the transition state conformation has an energy barrier ~2.5 times higher than the hydrolysis route. Moreover, the trapping of [alpha -32P]8-azido-ADP under non-hydrolysis conditions has an activation energy 1.5 times higher than that required for basal or verapamil-stimulated hydrolysis of ATP or 8-azido-ATP, which represents the complete catalytic cycle (see Table I and Fig. 10). This result is consistent with the hypothesis that the hydrolysis of [alpha -32P]8-azido-ATP provides energy to facilitate the conformational changes that accompany Vi-induced trapping. On the other hand, when [alpha -32P]8-azido-ADP is directly trapped into Pgp, there is no accompanying hydrolysis, and thus this reaction would necessarily have a much greater energy barrier to overcome; for this reason, in the normal catalytic cycle of ATP hydrolysis, this reaction would be highly unfavorable (see Fig. 10 for the comparison of the activation energies required for various steps in the catalytic cycle of ATP hydrolysis). These data also provide an explanation for previous studies (3), which show that the inhibition of ATPase activity at 37 °C was much more rapid with ATP and Vi than with ADP and Vi. Moreover, as shown in Table I, the activation energy for the binding of [alpha -32P]8-azido-ADP in the absence of Vi is only 19.5 kJ/mol. This is about 7.5-fold lower than the activation energy for the trapping of [alpha -32P]8-azido-ADP in the presence of Vi. This suggests that it is not the binding step but the subsequent conformational changes that generate Vi-trapped intermediate(s) that are energetically intensive. Similarly, the activation energy for the binding of the hydrophobic drug-substrate IAAP to Pgp is extremely low (7.97 kJ/mol).


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Fig. 10.   Activation energies (Eact) required for various steps in the catalytic cycle of ATP hydrolysis by Pgp. The arrows as indicated represent the values (kJ/mol) for the activation energies of nucleotide binding (empty arrow); Vi-induced trapping of [alpha -32P]8-azido-ADP through hydrolysis of [alpha -32P]8-azido-ATP (gray arrow); hydrolysis of ATP or 8-azido-ATP in the presence or absence of drug-substrate verapamil (hatched arrow); and Vi-induced [alpha -32P]8-azido-ADP trapping in the absence of hydrolysis (dark arrow). These data are from Fig. 9B and Table I, and only average or mean values are given here. See legend to Fig. 2A for the description of the catalytic scheme and the text for details.

In recent years, the crystal structures of the ATP subunits of several ABC and analogous transporters have been resolved. These include the following: HisP, the ATP subunit of the bacterial histidine permease (36); MutS, a protein that recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair (37, 38); ArsA, the soluble ATPase component of the bacterial arsenite pump, ArsAB (39); and MalK, the ATPase subunit of the trehalose/maltose transporter (40). In most of these studies (viz. MutS, ArsA, and MalK), the nucleoside diphosphate was directly incorporated by including ADP (and in some cases Vi or aluminum fluoride) during crystallization. Our study suggests that these structures where the ADP has not been incorporated in situ by the hydrolysis of ATP are nonetheless representative of the native conformation. Also, published reports postulate that the nucleoside diphosphate may have interesting regulatory roles to play in the catalytic cycles of several ABC transporters such as cystic fibrosis transmembrane conductance regulator and the sulfonyl urea receptor, SUR1 (41-43). In many of these instances [alpha -32P]8-azido-ADP would prove very useful in designing experimental strategies to address these hypotheses directly.

Taken together, our results provide compelling evidence that although, there is a 2.5-fold difference in the activation energies required to generate the Pgp·[alpha -32P]8-azido-ADP·Vi complex using [alpha -32P]8-azido-ADP and Vi compared with [alpha -32P]8-azido-ATP and Vi, the transition state complex generated by either route is functionally indistinguishable. Our preliminary observations with another ABC transporter, the MRP1 (44), suggest that MRP1 also exhibits Vi-induced trapping of [alpha -32P]8-azido-ADP under both hydrolysis and non-hydrolysis conditions.2 These findings are consistent with the results reported in this paper for Pgp. This work, however, does not address the effect of drug substrates on Vi-induced trapping of [alpha -32P]8-azido-ADP under non-hydrolysis conditions. These experiments are currently in progress.

    ACKNOWLEDGEMENTS

We thank Dr. Michael M. Gottesman for discussions, encouragement, and for critical comments on the manuscript. We also thank Drs. Christine A. Hrycyna and Michael M. Gottesman for providing the wild-type and mutant MDR1 constructs.

    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, Center for Cancer Research, NCI, Bldg. 37, Rm. 1B-22, National Institutes of Health, 37 Convent Dr., MD 20892-4255. Tel.: 301-402-4178; Fax: 301-435-8188; E-mail: ambudkar@helix.nih.gov.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M100886200

2 M. Muller, M. M. Smith, Z. E. Sauna, and S. V. Ambudkar, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MDR, multiple drug resistance; ABC, ATP-binding cassette; IAAP, [125I]iodoarylazidoprazosin; MES, 2[N-morpholino]ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; Pgp, P-glycoprotein; Vi, orthovanadate; TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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