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
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ABSTRACT |
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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 [ 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. [ 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 [ We demonstrate in this study that it is possible to initiate
Vi-induced trapping with either [ Chemicals--
[125I]IAAP, 2,200 Ci/mmol, was
obtained from PerkinElmer Life Sciences.
[ 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
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 Binding of [ Vanadate-induced [ 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.
Binding of [ Vi-induced Trapping of [
To characterize further Pgp in the transition state conformation
generated by using [ Effect of Divalent Cations on the Vi-induced Trapping of
[ Comparing the Kinetics of Generating the
Pgp·[ 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 [ Mutations in the Walker B Domain of Either the N- or
C-terminal ATP Sites of Pgp Arrest Vi-induced Trapping of
[ 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).
The Inhibition of IAAP Labeling of Pgp during Vi-induced Trapping
of 8-Azido-ADP and the Extent of Vi-induced Trapping of
[ Determination of the Activation Energies for Vi-induced Trapping
Using [
To understand this difference, we determined the activation energies
for these two processes. Fig. 9B depicts Arrhenius plots for
Vi-induced trapping using either [ 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, [ The results given in Fig. 1, A and B, indicate
that [ 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 [ 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
[ 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 [-32P]8-azido-ADP trapping into Pgp
in the presence of [
-32P]8-azido-ATP (with ATP
hydrolysis) or [
-32P]8-azido-ADP (without ATP
hydrolysis). Vi mimics Pi to trap the nucleotide
tenaciously in the Pgp·[
-32P]8-azido-ADP·Vi
conformation in either condition. Thus, by using [
-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
[
-32P]8-azido-ATP or
[
-32P]8-azido-ADP. The trapped
[
-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
[
-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 [
-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 [
-32P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the
Pgp·[
-32P]8-azido-ADP (or ADP)·Vi transition state
complexes generated either in the absence of or accompanying
[
-32P]8-azido-ATP hydrolysis are functionally indistinguishable.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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 [
-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.
-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 [
-32P]8-azido-ADP to Pgp. We
demonstrate that [
-32P]8-azido-ADP binds specifically
to Pgp with a Kd comparable to that for
[
-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.
-32P]8-azido-ADP or
[
-32P]8-azido-ATP, with similar kinetics, and that the
trapped [
-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 [
-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·[
-32P]8-azido-ADP·Vi
transition state complex starting with
[
-32P]8-azido-ADP is ~2.5 times greater than if
[
-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 [
-32P]8-azido-ADP should prove useful to
compare the catalytic cycle of ATP hydrolysis by different ABC transporters.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-Azido-ATP (15-20 Ci/mmol),
[
-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.
-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
-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).
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.
-32P]8-Azido-ATP or
[
-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
[
-32P]8-azido-ATP or
[
-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.
-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 [
-32P]8-azido-ATP or
[
-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
[
-32P]8-azido-ATP or
[
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-Azido-ADP to Pgp--
The
radiolabeled, photoaffinity analogue of ATP,
[
-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,
[
-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
[
-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
[
-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 [
-32P]8-azido-ADP is saturable with a
Kd of 10 ± 3 µM, which is very
similar to the Kd of
[
-32P]8-azido-ATP binding to Pgp, which is in the
range of 10-15 µM (12). These results suggest that
[
-32P]8-azido-ADP binds directly to Pgp in a manner
similar to [
-32P]8-azido-ATP.
View larger version (38K):
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Fig. 1.
[ -32P]8-azido-ADP binds
specifically to the ATP sites of Pgp. A, binding of
[
-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
[
-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, [
-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
[
-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 [
-32P]8-azido-ADP for Pgp
was 10 ± 3 µM.
-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
[
-32P]8-azido-ATP and Vi at 37 °C (i.e.
allowing [
-32P]8-azido-ATP to be hydrolyzed to
[
-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 [
-32P]8-azido-ADP
and 250 µM Vi for 10 min at 37 °C, the
[
-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
[
-32P]8-azido-ADP but in the absence of Vi, where
there is significantly less incorporation of
[
-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
[
-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 [
-32P]8-azido-ADP in the presence of
Vi. The autoradiogram also demonstrates that while the
[
-32P]8-azido-ADP incorporated in the absence of Vi
can be competed out by excess ATP (lane 2), the
[
-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
[
-32P]8-azido-ADP into Pgp suggesting that hydrolysis
is not a prerequisite for Vi-induced trapping per se but is
obligatory when [
-32P]8-azido-ATP (or any other
hydrolyzable nucleoside triphosphate) is used so as to generate the
nucleoside diphosphate.
View larger version (40K):
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Fig. 2.
[ -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[
-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, [
-32P]8-azido-ADP incubated with Pgp in
the presence of Vi results in [
-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 [
-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 [
-32P]8-azido-ADP; lane 2,
membranes treated with 50 µM
[
-32P]8-azido-ADP and 12.5 mM ATP was
added prior to UV cross-linking; lane 3, membranes treated
with 50 µM [
-32P]8-azido-ADP and 250 µM Vi; lane 4, membranes treated with 50 µM [
-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 [
-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
[
-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 [
-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
[
-32P]8-azido-ATP and Vi; lane 2, membranes
incubated with [
-32P]8-azido-ATP and Vi and treated
with trypsin; lane 3, control membranes incubated with
[
-32P]8-azido-ADP and Vi; and lane 4,
membranes incubated with [
-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).
-32P]8-azido-ADP, we compared the
distribution of the trapped [
-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 [
-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
[
-32P]8-azido-ADP is generated through the hydrolysis
of [
-32P]8-azido-ATP or directly providing the
nucleoside diphosphate itself.
-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 [
-32P]8-azido-ADP (11, 27, 31). Compared
with the extent of Vi-induced [
-32P]8-azido-ADP
trapping in the presence of Mg2+, Mn2+, and
Co2+, the amount of [
-32P]8-azido-ADP
incorporated in the presence of Ca2+ was negligible (31).
In Fig. 3 we compared the Vi-induced
trapping of [
-32P]8-azido-ADP under hydrolysis
conditions (i.e. initiating the reaction with
[
-32P]8-azido-ATP and Vi) and non-hydrolysis
conditions (i.e. initiating the reaction with
[
-32P]8-azido-ADP and Vi). We find that
Mg2+, Mn2+, and Co2+ permit
Vi-induced trapping of [
-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 [
-32P]8-azido-ADP even in the
absence of hydrolysis.
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Fig. 3.
Effect of divalent cations on the
incorporation of
[ -32P]8-azido-ADP into Pgp
during Vi-induced trapping. Crude membranes were incubated with 50 µM [
-32P]8-azido-ATP or
[
-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
[
-32P]8-azido-ADP incorporated when trapping is
carried out under hydrolysis conditions by incubation with
[
-32P]8-azido-ATP and Vi and the empty bars
represent nucleotide trapping carried out under non-hydrolysis
conditions by incubation with [
-32P]8-azido-ADP and
Vi.
-32P]8-Azido-ADP·Vi Transition State
Intermediates Using Either [
-32P]8-Azido-ATP or
[
-32P]8-Azido-ADP and Vi--
We have shown above
that it is possible to generate the
Pgp·[
-32P]8-azido-ADP·Vi transition state
intermediate by directly incubating Pgp with
[
-32P]8-azido-ADP and Vi. It would be important to
determine the kinetics of Vi-induced [
-32P]8-azido-ADP
trapping under hydrolysis and non-hydrolysis conditions. The
kinetics of trapping using [
-32P]8-azido-ATP + Vi or
[
-32P]8-azido-ADP + Vi are depicted in Fig.
4, A and B. The
incorporation of [
-32P]8-azido-ADP through the
hydrolysis of increasing concentrations of
[
-32P]8-azido-ATP or in the presence of increasing
concentrations of [
-32P]8-azido-ADP itself exhibits
Michaelis-Menten kinetics with a Km of 20 ± 4.3 µM for [
-32P]8-azido-ATP and a
Kd of 16 ± 1.6 µM for
[
-32P]8-azido-ADP, respectively. Thus, the affinity of
[
-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
[ -32P]8-azido-ADP into Pgp under
hydrolysis and non-hydrolysis conditions. A, kinetics
of [
-32P]8-azido-ADP trapping into Pgp under
hydrolysis conditions by using [
-32P]8-azido-ATP and
Vi. Crude membranes (1 mg/ml) were incubated in the ATPase assay buffer
containing increasing concentrations of
[
-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 [
-32P]8-azido-ATP for Pgp in the presence of Vi was
20 ± 4.3 µM. B, kinetics of
[
-32P]8-azido-ADP trapping into Pgp using
[
-32P]8-azido-ADP and Vi. Crude membranes (2 mg/ml)
were incubated in the ATPase assay buffer containing increasing
concentrations of [
-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
[
-32P]8-azido-ADP for Pgp in the presence of Vi was
16 ± 1.6 µM.
-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.
-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·[
-32P]8-azido-ADP·Vi catalytic state
intermediate by directly incubating Pgp with
[
-32P]8-azido-ADP and Vi, and the kinetics are
comparable regardless of whether [
-32P]8-azido-ADP or
[
-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 [
-32P]8-azido-ADP generated
through the hydrolysis of [
-32P]8-azido-ATP (6, 9). If
only ATP hydrolysis is affected in the mutants, using
[
-32P]8-azido-ADP instead of
[
-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
[
-32P]8-azido-ATP or
[
-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
[
-32P]8-azido-ADP regardless of whether it is
generated through hydrolysis of [
-32P]8-azido-ATP or
when [
-32P]8-azido-ADP is provided to Pgp.
Additionally, neither mutant supports Vi-induced trapping of
[
-32P]8-azido-ADP with either
[
-32P]8-azido-ATP or
[
-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
[ -32P]8-azido-ADP using either
[
-32P]8-azido-ADP or
[
-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
[
-32P]8-azido-ATP or
[
-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
[
-32P]8-azido-ADP under hydrolysis (lanes
1-3) and non-hydrolysis conditions (lanes 4-6).
Lane 1, wild-type Pgp membranes labeled with
[
-32P]8-azido-ATP; lane 2, D555N membranes
labeled with [
-32P]8-azido-ATP; lane 3, D1200N membranes labeled with [
-32P]8-azido-ATP;
lane 4, wild-type membranes labeled with
[
-32P]8-azido-ADP; lane 5, D555N membranes
labeled with [
-32P]8-azido-ADP; and lane 6,
D1200N membranes labeled with [
-32P]8-azido-ADP. An
equal amount of crude membranes (60 µg of protein) was
immunoprecipitated and loaded into each lane.
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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.
-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 [
-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
[
-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 [
-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
[ -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 [
-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 [
-32P]8-azido-ADP (
) into Pgp.
Inset compares the extent of
[
-32P]8-azido-ADP and IAAP incorporated into Pgp at
each time point; the two are inversely correlated (r = 0.92).
-32P]8-Azido-ATP and
[
-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
[
-32P]8-azido-ADP. There is a low level of
[
-32P]8-azido-ADP trapping at 23 °C, which is
significantly increased at 37 °C, when Pgp is incubated with
[
-32P]8-azido-ATP and Vi for 6 min. However, when
[
-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
[
-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
[ -32P]8-azido-ADP initiated with
[
-32P]8-azido-ATP or
[
-32P]8-azido-ADP.
A, Vi-induced trapping of [
-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
[
-32P]8-azido-ATP (2-4 µCi/nmol) or
[
-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 [
-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 [
-32P]8-azido-ATP (4 µCi/nmol) or
[
-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 [
-32P]8-azido-ADP
incorporated) was used to calculate the activation energy as
described in the legend to Table I for Vi-induced
[
-32P]8-azido-ADP trapping into Pgp initiated by using
[
-32P]8-azido-ATP (
) or
[
-32P]8-azido-ADP (
).
-32P]8-azido-ADP or
[
-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 [
-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
[
-32P]8-azido-ADP and Vi even at 23 °C was
undetectable (cf. Fig. 9A). Thus, if
[
-32P]8-azido-ADP is trapped into Pgp by incubating
[
-32P]8-azido-ATP and Vi, allowing the
[
-32P]8-azido-ATP to be hydrolyzed to
[
-32P]8-azido-ADP, which is then trapped, the energy
barrier is significantly lower than if
[
-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
[
-32P]8-azido-ADP under hydrolysis and non-hydrolysis
conditions, and for the binding of [
-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 [
-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
[
-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
[
-32P]8-azido-ADP or
[
-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).
Activation energies (Eact) for basal and verapamil-stimulated
ATP hydrolysis, 8-azido-ATP hydrolysis, Vi-induced
[-32P]8-azido-ADP trapping, [
-32P]8-azido-ADP
binding, and IAAP binding
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-azido-ATP. The
Pgp·[
-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, [
-32P]8-azido-ADP, under
both hydrolysis and non-hydrolysis conditions.
-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 [
-32P]8-azido-ADP
in the presence of Vi is trapped into the
Pgp·[
-32P]8-azido-ADP·Vi transition state. This
experiment clearly distinguishes [
-32P]8-azido-ADP
that is trapped in the ternary,
Pgp·[
-32P]8-azido-ADP·Vi, complex from binding of
nucleotide per se. It is clear that in the absence of Vi,
[
-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, [
-32P]8-azido-ADP is
tenaciously trapped in the
Pgp·[
-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 [
-32P]8-azido-ATP in the
presence of Vi, [
-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
[
-32P]8-azido-ADP into the N- and C-terminal halves
regardless of whether the [
-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 [
-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 [
-32P]8-azido-ADP under both
hydrolysis and non-hydrolysis conditions.
-32P]8-azido-ADP via
the hydrolysis of [
-32P]8-azido-ATP into Pgp, the
Km for [
-32P]8-azido-ATP is nearly
identical to the Kd of
[
-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
[
-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, [
-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 [
-32P]8-azido-ADP generated
through the hydrolysis of [
-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
[
-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.
-32P]8-azido-ADP trapping and inhibition of IAAP
binding during Vi-induced trapping of
[
-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).
-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
[
-32P]8-azido-ADP trapping at 23 °C. Arrhenius
plots of the Vi-induced trapping initiated by either
[
-32P]8-azido-ATP or
[
-32P]8-azido-ADP show that these pathways have
different activation energies (Fig. 9B). When Vi-induced
trapping is effected by incubating [
-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
[
-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
[
-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 [
-32P]8-azido-ATP
provides energy to facilitate the conformational changes that accompany
Vi-induced trapping. On the other hand, when
[
-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 [
-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 [
-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).
View larger version (15K):
[in a new window]
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
[ -32P]8-azido-ADP through hydrolysis of
[
-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
[
-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 [-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·[-32P]8-azido-ADP·Vi complex
using [
-32P]8-azido-ADP and Vi compared with
[
-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
[
-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 [
-32P]8-azido-ADP
under non-hydrolysis conditions. These experiments are currently in progress.
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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.
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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.
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.
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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.
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