From the Laboratory of Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, December 14, 2000, and in revised form, January 10, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
P-glycoprotein (Pgp) is a plasma membrane protein
whose overexpression confers multidrug resistance to tumor cells by
extruding amphipathic natural product cytotoxic drugs using the energy
of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our
understanding of the mechanism of ATP-binding cassette transporters. We
have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520)
that there are two independent ATP hydrolysis events in a single
catalytic cycle of Pgp. In this study we exploit the vanadate
(Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to
address the question of what are the effects of ATP hydrolysis on the
nucleotide-binding site. We find that at the end of the first
hydrolysis event there is a drastic decrease in the affinity of
nucleotide for Pgp coincident with decreased substrate binding. Release
of occluded dinucleotide is adequate for the next hydrolysis event to
occur but is not sufficient for the recovery of substrate binding.
Whereas the two hydrolysis events have different functional outcomes
vis à vis the substrate, they show comparable
t1/2 for both incorporation and release of
nucleotide, and the affinities for [ Expression of the plasma membrane protein, P-glycoprotein
(Pgp),1 confers multidrug
resistance to tumor cells. It belongs to the ATP-binding cassette (ABC)
superfamily of transport proteins, characterized by two homologous
halves containing six transmembrane helices and one nucleotide-binding
site in each half (1, 2). Pgp confers drug resistance to tumor cells by
extruding cytotoxic natural product hydrophobic drugs using the energy
of ATP hydrolysis (2, 3). Pgp interacts not only with nucleotides
(4-6) and cytotoxic drugs (3) but also with a diverse set of other
lipophilic compounds (7). Additionally, besides the transport of these compounds coupled to ATP hydrolysis, other complex interactions are
known to occur in Pgp with modulators of the multidrug resistance (MDR)
phenotype (8). Considering the importance of Pgp in cancer chemotherapy
and as a model system for ABC transporters in general, a clear
understanding of the catalytic cycle of this transporter is of
considerable importance.
The first catalytic scheme proposed for Pgp (9) was that of the ATP
hydrolysis reaction. The essential feature of this model is alternating
hydrolysis of ATP at the two ATP-binding sites. It was postulated that
nucleotide first binds to one of the two sites but could not be
hydrolyzed. When another nucleotide binds to the second site it
promotes hydrolysis at the first site, which in turn powers substrate
transport. In the next cycle, hydrolysis occurs at the second ATP site.
We have recently (10) demonstrated that there is substantially greater
complexity in the catalytic cycle of Pgp. We simultaneously monitored
changes in the substrate- and nucleotide-binding sites to show that
although binding of nucleotide per se does not affect
interactions with the substrate, ATP hydrolysis results in a dramatic
conformational change where the affinity of the substrate
[125I]iodoarylazidoprazosin ([125I]IAAP)
for Pgp trapped in the transition state conformation (Pgp·ADP·Vi) is reduced >30-fold. Even more remarkable is the finding that to
transform Pgp from this intermediate state of low affinity for
substrate to the next catalytic cycle, i.e. a conformation that binds substrate with high affinity, the hydrolysis of an additional molecule of nucleotide is obligatory. This requirement for
two ATP hydrolysis events in the course of a single catalytic cycle was
consistent with the measured stoichiometry for Pgp where two ATP
molecules are hydrolyzed for each substrate molecule transported (11,
12).
In this study we have analyzed, in real time, the repeating succession
of vanadate (Vi)-induced trapping and release of
[ Chemicals--
Cyclosporin A was purchased from Calbiochem. and
[125I]Iodoarylazidoprazosin ([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 (H6)) as described (13). Crude membranes were prepared as
described previously (4, 13).
Purification of Pgp--
Human Pgp was purified as described
previously (13) with minor changes. Briefly, crude Pgp-containing
membranes were solubilized with octyl- Photoaffinity Labeling of Pgp with
[125I]IAAP--
The crude membranes (10-50 µg) were
incubated with the drug or modulator for 3 min at room temperature in
50 mM Tris-HCl, pH 7.5, and [125I]IAAP
(unless otherwise stated, 3-6 nM) was added and incubated for an additional 5 min under subdued light. The samples were then
illuminated with a UV lamp assembly (PGC Scientifics, Gaithersburg, MD)
fitted with two black light (self-filtering) UV-A long wave F15T8BLB
tubes (365 nm) for 10 min at room temperature (21-23 °C). Following
SDS-PAGE on a 8% Tris glycine gel at constant voltage, gels were dried
and exposed to Bio-Max MR film (Eastman Kodak Co.) at Vanadate-induced [ Binding of [ Binding of TNP-ATP to Pgp--
Binding of the fluorescent
analogue TNP-ATP was measured by determining the increase in
fluorescence signal of TNP-ATP in the presence of purified Pgp
incorporated into proteoliposomes. Proteoliposomes (20-25 µg of
protein) were incubated with 100 µM TNP-ATP in ATPase
assay buffer in a reaction volume of 200 µl for 10 min at room
temperature (22-23 °C) under subdued light. The samples were then
transferred to individual wells in a 96-well flat bottom,
UV-transparent, disposable microplate (Spectraplate, Molecular Devices,
Sunnyvale, CA) and an emission scan (500-600 nm) obtained using
excitation at 408 nm on a Spectra MAX 250 Microplate spectrofluorimeter
(Molecular Devices, Sunnyvale, CA). The enhancement in fluorescence due
to TNP-ATP binding was estimated by running an emission scan of TNP-ATP
in the assay buffer in the absence of Pgp. Additionally, to obtain
signal specific to Pgp, an equivalent emission scan in the presence of
10 mM ATP (i.e. 100-fold excess) was generated
and subtracted from the scan obtained in the absence of ATP.
The Vi-induced ADP-trapped Conformation of Pgp during ATP
Hydrolysis Exhibits a Marked Decrease in Affinity for Both Drug
Substrate [125I]IAAP and
[
To quantify the decrease in affinity for nucleotide in the
Pgp·8-azido-ADP·Vi complex, we estimated the apparent
Kd value of 8-azido-ATP for Pgp before and after
Vi-induced trapping. Fig. 2C depicts that
[ The Vi-induced ADP-trapped Conformation of Pgp during ATP
Hydrolysis Exhibits a Marked Decrease in Affinity for the Fluorescent
Nucleotide TNP-ATP--
To exclude the possibility that the reduced
binding of nucleotide to Pgp in the transition state is an artifact of
photoaffinity cross-linking, another approach using the fluorescent
nucleotide analogue TNP-ATP was used. TNP-ATP is a hydrolyzable,
fluorescent analogue of ATP (17). Both Chinese hamster and human Pgp
can hydrolyze TNP-ATP but at a much slower rate than ATP, and TNP-ATP has also been used to characterize the ATP sites on Pgp (18, 19). The
compound exhibits a low quantum yield in an aqueous medium with an
emission maxima at 550 nm (excitation 408 nm), and an increase in
quantum yield accompanied by a small blue shift (new emission maxima at
530 nm) occurs when TNP-ATP binds to the ATP sites of Pgp (19).
However, when Pgp is pretreated with Vi and 8-azido-ATP at 37 °C to
generate the Pgp·8-azido-ADP·Vi complex, there is a marked decrease
in the fluorescence (Fig. 3A)
suggesting that the binding of nucleotide (TNP-ATP) to Pgp is
drastically reduced, consistent with the results obtained in the
experiments with [
The kinetics of inhibition of TNP-ATP binding to Pgp in the presence of
ATP and Vi are depicted in Fig. 3B. Purified, reconstituted Pgp was treated with increasing concentrations of ATP in the presence of Vi at 37 °C for 10 min, and excess ATP and Vi were removed by
centrifugation. Extent of TNP-ATP binding to Pgp was quantified by
measuring the fluorescence intensity (Ex = 408 nm and Em = 530 nm). A single phase exponential decay model fits the data with a
Ki(ATP) of 83 µM.
Dissociation of [
To address the second question, i.e. what controls the
release of ADP from Pgp after ATP hydrolysis, we monitored the
dissociation of [ Characterization of the Two ATP Hydrolysis Events in a Single
Catalytic Cycle of Pgp--
The experiments described above
demonstrate that the ATP hydrolysis that follows binding of nucleotide
and substrate results in a conformational change in Pgp that
dramatically lowers the affinity of both substrate and nucleotide and
inhibits the binding of additional molecules. Fig.
5A depicts an experimental
strategy, described in detail in the legend, to discriminate between
the two hydrolysis events postulated to occur in a single catalytic cycle of Pgp (10). Essentially, Pgp can be put through repeating cycles
of Vi-induced [ Binding of [125I]IAAP to Pgp at Various Steps in the
Catalytic Cycle of ATP Hydrolysis--
We postulated earlier that two
hydrolysis events occur during a single cycle of Pgp (10) based on the
following evidence: (a) following ATP hydrolysis Pgp takes
on a conformation with >30-fold reduced affinity for substrate, and
(b) for Pgp to regain the conformation that binds substrate
with high affinity a second ATP hydrolysis event is mandatory. Although
Fig. 5B tracks ATP hydrolysis over the entire catalytic
cycle and is consistent with our previous findings, it does not
explicitly demonstrate the status of the substrate-binding site prior
to and following each hydrolysis event. Fig.
6 shows [125I]IAAP binding
to Pgp at different steps in the catalytic cycle as depicted in Fig.
5B, which, re-plotted on a continuous time scale, is shown
as a gray backdrop. The gray area thus represents the extent of [
Prior to the first hydrolysis event corresponding to the zero time
point in Fig. 5A (I), there is a normal level of
[125I]IAAP binding (Fig. 6, step I), similar
to untreated Pgp. Immediately following ATP hydrolysis, i.e.
the final time point in Fig. 5A (step I) or the
first time point in Fig. 5A (step II), the
[125I]IAAP binding is reduced by >90% (Fig. 6,
step II). Following release of occluded 8-azido-ADP, in the
absence of ATP hydrolysis (the final time point in Fig. 5A
(step II)), [125I]IAAP binding
continues to be reduced (Fig. 6, step III).
Vi-induced trapping of 8-azido-ADP in the second cycle (Fig.
5A (step III)) does not show any recovery of
[125I]IAAP binding (Fig. 6, step
IV). However, instead of trapping 8-azido-ADP, if hydrolysis
is allowed to occur in the absence of Vi, there is full recovery of
[125I]IAAP binding (Fig. 6, step Va).
Similarly, release of the occluded 8-azido-ADP following the second
hydrolysis (Fig. 5A (step V)) allows recovery of
[125I]IAAP binding (Fig. 6, step V). Taken
together with Fig. 5B, these results further support the
model we have previously proposed (10) and establish that two
kinetically similar but functionally different ATP hydrolysis events
are occurring in a single catalytic cycle.
The Two Hydrolysis Events in a Single Catalytic Cycle of Pgp Have
Identical Km Values for [ Our recent work has demonstrated that there are two independent
ATP hydrolysis events in a single catalytic cycle of Pgp. In this
study, we exploit the vanadate (Vi)-induced transition state
conformation of Pgp (Pgp·ADP·Vi) to address the question of what
are the effects of ATP hydrolysis on the nucleotide-binding site. We
also track, in real time, the repeating succession of trapping and
release of [ Implicit in the model for the catalytic cycle proposed by Senior and
others (24, 26, 27) is long range conformational coupling between the
drug- and nucleotide-binding sites. Although direct evidence for such a
relationship was lacking, interactions such as the stimulation of
ATPase activity by drugs (4, 5, 30) have been accepted as evidence for
the structural interactions that underlie such a coupling (26, 27). Our
recent work (10) and Fig. 2A on the other hand
quantitatively corroborate that large changes occur in the
substrate-binding site as a consequence of ATP hydrolysis. It is
critical, however, to demonstrate that these effects are not
coincidental. A comparison of the Km for Vi-induced
trapping of [ The consequences of ATP hydrolysis on substrate binding have been
elucidated elsewhere (10) and are described briefly above. To
understand the effect of ATP hydrolysis on the affinity of nucleotides
for the ATP sites, we once more exploited the Vi-trapped, transition
state complex (Pgp·ADP·Vi). Fig. 2C illustrates that if crude membranes containing Pgp are pretreated with 8-azido-ATP and
Vi, they show reduced binding of [ There is strong evidence to support the occurrence of two hydrolysis
events in a single catalytic cycle. Useful information should be gained
by studying these two events independently. Data depicted in Fig.
5B, which follows the experimental strategy depicted in Fig.
5A, show that it is experimentally possible to propel Pgp
through repeating cycles of Vi-induced
[ Earlier studies with plasma membranes from Chinese hamster ovary cells
overexpressing Pgp clearly showed that the trapped [ On the basis of this study, we propose a model for the catalytic cycle
of Pgp, which is an extension of the one we proposed recently (10), and
this is illustrated in Fig. 8. The drug
and ATP first bind to Pgp (step I), because there is no
energetic requirement for this step (36). Additionally, drugs do not
affect nucleotide binding (Fig. 1) nor do nucleotides influence the
binding of substrate. The binding of nucleotide and drug is followed by the first hydrolysis event (step II), which is accompanied
by a conformational change that reduces the affinity of both substrate (Ref. 10 and Fig. 2A) and nucleotide (Figs. 2C
and 3A) for Pgp. This intermediate can be trapped by using
Vi, an analogue of Pi that generates the stable
Pgp·ADP·Vi complex (step IIIA). Following hydrolysis,
ADP is released (step IV). This release occurs spontaneously and is not influenced by the presence of nucleotides (Fig.
4B). The dissociation of ADP is accompanied by a
conformational change that allows nucleotide binding (Fig.
5B) but substrate binding continues to be reduced (Fig. 6).
A second ATP hydrolysis event is then initiated (step V)
which is kinetically indistinguishable from the first (Fig. 7), at
which point the substrate binding is still not regained (Fig. 6). This
event too can be captured as an intermediate using Vi to trap the
nucleotide (steps VIA and VIB). The subsequent
release of ADP (step VII) completes one catalytic cycle
bringing the Pgp molecule back to the original state where it can bind
both substrate and nucleotide to initiate the next cycle (Fig. 6). This
revised model thus incorporates key elements based on the work reported
here to the scheme that we proposed earlier (10). The conformation of
Pgp, following ATP hydrolysis, shows reduced affinity for the
nucleotide (steps II, III, IIIA, V, VI, and VIA).
Additionally, following the second ATP hydrolysis event, the release of
ADP from Pgp is essential to complete the catalytic cycle,
i.e. to bring the molecule back to the state where it will
bind the next molecule of drug substrate (steps VIB and
VII). Finally, this model is consistent with our recent
finding that ADP release at steps IV and VII (16) appear to be the
rate-limiting steps in the catalytic cycle.
-32P]8-azido-ATP
during Vi-induced trapping are identical. In addition, the
incorporation of [
-32P]8-azido-ADP in two ATP sites
during both hydrolysis events is also similar. These data demonstrate
that during individual hydrolysis events, the ATP sites are recruited
in a random manner, and only one site is utilized at any given time
because of the conformational change in the catalytic site that
drastically reduces the affinity of the second ATP site for nucleotide
binding. In aggregate, these findings provide an explanation for the
alternate catalysis of ATP hydrolysis and offer a mechanistic framework
to elucidate events at both the substrate- and nucleotide-binding sites
in the catalytic cycle of Pgp.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-azido-ADP through an entire catalytic cycle,
and we monitored the substrate ([125I]IAAP) binding at
the beginning and end of each trapping (hydrolysis) event. The results,
besides validating our model, provided experimental strategies for
independently studying the kinetics of the two hydrolysis events. We
find that at the end of the first hydrolysis event there is a >30-fold
decrease in the affinity of nucleotide for Pgp coincident with the
decreased substrate binding shown earlier (10, 13). Release of occluded
ADP or 8-azido-ADP is adequate for the next hydrolysis event to occur
but is not sufficient for recovery of substrate binding, which occurs
only at the end of the second hydrolysis, i.e. after ADP
dissociates from the transporter. Whereas the two hydrolysis events
have different functional outcomes vis à vis the
substrate, they show comparable t1/2 for both
incorporation and release of nucleotide, and the Km
values for [
-32P]8-azido-ATP during the Vi-induced
trapping during both hydrolysis events are identical. Similarly, the
incorporation of [
-32P]8-azido-ADP in both the
N-terminal and the C-terminal ATP sites is identical during both
ATP hydrolysis events. These data demonstrate that during individual
hydrolysis events the ATP sites are recruited in a random manner, and
the fact that only one site is utilized at any given time is a
consequence of the conformational change that reduces the affinity of
nucleotide for the second ATP site. Thus, the prevention of ATP binding
to the second site while the first one is in a catalytic conformation
appears to be the explanation for alternate catalysis in Pgp.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-Azido-ATP (15-20 Ci/mmol) and 8-azido-ATP
were purchased from Affinity Labeling Technologies, Inc (Lexington,
KY). TNP-ATP was procured from Molecular Probes (Eugene OR). All other
chemicals were obtained from Sigma.
-D-glucopyranoside
(1.25%) in the presence of 20% glycerol and lipid mixture (0.1%).
Solubilized proteins were subjected to metal affinity chromatography
(Talon resin, CLONTECH, Palo Alto, CA) in the
presence of 0.95% octyl-
-D-glucopyranoside and 0.04%
lipid; 80% purified Pgp was eluted with 100 mM imidazole. Pgp in 100 mM imidazole fraction was then concentrated
(Centriprep-50, Amicon, Beverly, MA) to ~0.5 mg/ml and stored at
70 °C. Pgp was identified by immunoblot analysis using the
monoclonal antibody C219 (13) and quantified by Amido Black protein
estimation method as described previously (14).
70 °C for
12-24 h. The radioactivity incorporated into the Pgp band was
quantified using the STORM 860 PhosphorImager system (Molecular
Dynamics, Sunnyvale, CA) and the software ImageQuaNT.
-32P]8-Azido-ADP Trapping in
Pgp--
The Pgp·ADP·Vi or Pgp·8-azido-ADP·Vi transition state
conformation was generated as described previously with minor
modifications (10). Crude membranes (1 mg/ml) were incubated in the
ATPase assay buffer (40 mM MES-Tris, pH 6.8, 50 mM KCl, 5 mM sodium azide, 2 mM
EGTA, 2 mM dithiothreitol, and 10 mM
MgCl2) containing 50 µM
[
-32P]8-azido-ATP (containing 4 µCi/nmol) and 250 µM Vi in the dark at 37 °C for 5 min. The reaction was
stopped by the addition of 12.5 mM ice-cold ATP and placing
the sample immediately on ice. The trapped nucleotides were
photo-cross-linked as described below.
-32P]8-Azido-ATP to Pgp--
Crude
membranes (1 mg/ml) were incubated in the ATPase assay buffer
containing 10 µM [
-32P]8-azido-ATP
(containing 2-5 µCi/nmol) in the dark at 4 °C for 5 min. The
samples were then irradiated with an UV lamp assembly (365 nm) for 10 min on ice (4 °C). Ice-cold ATP (12.5 mM) was added to
displace excess noncovalently bound [
-32P]8-azido-ATP.
Excess nucleotides were removed by centrifugation at 300,000 × g at 4 °C for 10 min by using S120-AT2 rotor in a RC-M120EX micro-ultracentrifuge (Sorvall, Newtown, CT), and the pellet
was resuspended in 1× SDS-PAGE sample buffer. Following SDS-PAGE on an
8% Tris glycine gel at constant voltage, gels were dried and exposed
to Bio-Max MR film at
70 °C for 12-24 h. The radioactivity
incorporated into the Pgp band was quantified using the STORM 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the
software ImageQuaNT.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-Azido-ATP--
8-Azido-ATP is a good
hydrolysis substrate for both Chinese hamster and human Pgp, with a
Km similar to ATP (6, 15, 16).
-32P-Labeled 8-azido-ATP is a useful photoaffinity
reagent to understand the nucleotide binding and ATP hydrolysis events
that accompany drug-substrate transport in Pgp. Our recent studies (10)
show that binding of nucleotide to Pgp per se does not
affect its interactions with the substrate, similarly Fig.
1 demonstrates that the binding of the
nucleotide, [
-32P]8-azido-ATP, is not affected by
substrates such as verapamil and cyclosporin A. Earlier work from our
laboratory (10, 13) demonstrated that the Vi-trapped conformation of
Pgp binds substrates with at least 30-fold reduced affinity. The data
in Fig. 2A show that if the
photoaffinity substrate analogue, [125I]IAAP, is allowed
to bind Pgp, which has been treated previously with increasing
concentrations of 8-azido-ATP in the presence of Vi at 37 °C (which
corresponds to increased trapping of 8-azido-ADP in Pgp), there is a
concentration-dependent decrease in substrate binding. A
single phase exponential decay model fits the data with a
Ki = 18 µM for the 8-azido-ATP
inhibition of [125I]IAAP binding. It is also clear from
these data that for maximal trapping of 8-azido-ADP in the presence of
Vi, 0.5 to 1 mM 8-azido-ATP is required. Our earlier
work (10) and data in Fig. 2A clearly show that the
Pgp·8-azido-ADP·Vi conformation of Pgp exhibits a large decrease in
affinity for substrate. This raises the question as to how nucleotide
binding to Pgp is affected in the Vi-trapped state (i.e.
transition state). To address this question we generated the
Pgp·8-azido-ADP·Vi complex in the presence of increasing
concentrations of 8-azido-ATP and Vi, washed off excess nucleotide and
Vi by centrifugation, and then quantified the extent of
[
-32P]8-azido-ATP binding to Pgp. The results of this
experiment are graphically depicted in Fig. 2B. The
Vi-induced 8-azido-ADP-trapped conformation of Pgp shows a
concentration-dependent decrease in binding of nucleotide,
similar to that seen for substrate (Fig. 2A). A single phase
exponential decay model fits the data, and the Ki of
8-azido-ATP (during trapping) for the inhibition of nucleotide binding
is 12 µM and is comparable to the Ki of 8-azido-ATP (during trapping) for the inhibition of
[125I]IAAP binding which is 18 µM.
View larger version (99K):
[in a new window]
Fig. 1.
Effect of substrates on the binding of
[ -32P]8-azido-ATP to Pgp.
Crude membranes (20 µg of protein) were incubated in the ATPase assay
buffer with Me2SO, 50 µM verapamil, or
10 µM cyclosporin A at 37 °C for 10 min. The samples
were transferred to ice, and 10 µM
[
-32P]8-azido-ATP (containing 4 µCi/nmol) was added
under subdued lighting, incubated at 4 °C for 5 min, and then
UV-irradiated (365 nm) for 10 min on ice (4 °C). Ice-cold ATP (12.5 mM) was added to displace noncovalently bound
[
-32P]8-azido-ATP, and excess nucleotides were removed
by centrifugation at 300,000 × g at 4 °C for 10 min. The pellet was resuspended in 1× SDS-PAGE sample buffer, and
following SDS-PAGE the gels were dried and exposed to x-ray film at
70 °C for 12-24 h. Autoradiogram shows untreated Pgp (lane
1); Pgp pretreated with 50 µM verapamil (lane
2), or 10 µM cyclosporin A (lane
3).
View larger version (10K):
[in a new window]
Fig. 2.
The Pgp·8-azido-ADP·Vi transition state
complex shows reduced affinity for both [125I]IAAP and
[ - 32P]8-azido-ATP.
A, [125I]IAAP binding to Pgp pretreated with
8-azido-ATP and Vi. Crude membranes (20 µg of protein) were treated
with increasing concentrations of 8-azido-ATP (1-1000
µM) and Vi (250 µM) at 37 °C for 10 min
in the dark. Excess 8-azido-ATP and Vi were removed by centrifugation
at 300,000 × g at 4 °C for 10 min. The membranes
were resuspended in 50 mM Tris-HCl, pH 7.5, and labeled
with 5 nM [125I]IAAP as described under
"Experimental Procedures." [125I]IAAP incorporated in
the Pgp band was quantified using a PhosphorImager. B,
[
-32P]8-azido-ATP binding to Pgp pretreated with
8-azido-ATP and Vi. Crude membranes (20 µg of protein) were treated
with increasing concentrations of 8-azido-ATP and Vi as described in
A. Excess 8-azido-ATP and Vi were removed by centrifugation
at 300,000 × g at 4 °C for 10 min. The membranes
were resuspended in ATPase assay buffer and labeled with 10 µM [
-32P]8-azido-ATP (4 µCi/nmol) at
4 °C, as described in "Experimental Procedures."
[
-32P]8-Azido-ATP incorporated in the Pgp band was
quantified using a PhosphorImager. Inset shows the Pgp band
labeled with [
-32P]8-azido-ATP that has been
quantified in the graph. C, determination of the affinity of
Pgp for [
-32P]8-azido-ATP before and after Vi-induced
8-azido-ADP trapping. Crude membranes (20 µg of protein) were
incubated with increasing concentrations of
[
-32P]8-azido-ATP (5-10 µCi/nmol) at 4 °C, as
described under "Experimental Procedures." Excess nucleotide was
removed by centrifugation at 300,000 × g at 4 °C
for 10 min. The membranes were resuspended in 1× SDS-PAGE buffer and
electrophoresed on an 8% Tris glycine gel.
[
-32P]8-Azido-ATP incorporated in the Pgp band was
quantified using a PhosphorImager. Figure shows binding of
[
-32P]8-azido-ATP to control (
) membranes and those
pretreated with 1.25 mM 8-azido-ATP and 250 µM Vi at 37 °C for 10 min as described in B
to trap Pgp in the transition state conformation (
).
-32P]8-azido-ATP binding to control membrane vesicles
shows saturation; a hyperbola fits the data suggesting a single binding
site or more than one site with the same affinity, and the
Kd of [
-32P]8-azido-ATP was
computed to be 13 µM. Pgp previously trapped with Vi (250 µM) and 8-azido-ATP (1.2 mM) on the other
hand shows greatly reduced binding of
[
-32P]8-azido-ATP, which is not saturable even at a
concentration of 100 µM (data not shown). This suggests a
large decrease (>30-fold) in the affinity of nucleotide for Pgp in the
Vi-trapped conformation.
-32P]8-azido-ATP (Fig. 2,
B and C).
View larger version (8K):
[in a new window]
Fig. 3.
The Pgp·8-azido-ADP·Vi transition state
complex shows reduced binding of the fluorescent nucleotide
TNP-ATP. A, TNP-ATP binding to Pgp in transition state
conformation generated by pretreatment with 8-azido-ATP and Vi.
Purified Pgp was reconstituted into liposomes as described previously
(16). Proteoliposomes (25 µg of protein) were incubated with 100 µM TNP-ATP at room temperature for 10 min, and emission
scans were obtained (excitation = 408 nm; emission = 500-600
nm). Scans taken in the presence of Pgp were corrected for nonspecific
binding by subtracting scans acquired in the presence of 100-fold
excess ATP. Figure shows TNP-ATP + Pgp, control (continuous
line) and TNP-ATP + Pgp pre-trapped with 8-azido-ADP and Vi
(dashed line). Pgp was trapped with 8-azido-ADP by
incubating with 8-azido-ATP, 1.25 mM in the presence of Vi,
0.25 mM for 10 min at 37 °C. The reaction was stopped by
the addition of 10 mM ice-cold ATP, and excess nucleotides
and Vi were removed by centrifugation. B, kinetics of
inhibition of TNP-ATP binding to Pgp pretreated with ATP and Vi.
Proteoliposomes containing purified Pgp (20-25 µg) were treated with
increasing concentrations of ATP and Vi (250 µM) at
37 °C for 10 min. Excess ATP and Vi were removed by centrifugation
at 300,000 × g at 4 °C for 10 min. The membranes
were resuspended in ATPase assay buffer and incubated with 100 µM TNP-ATP at room temperature for 10 min, and emission
scans were obtained (excitation = 408 nm; emission = 500-600
nm). Scans taken in the presence of Pgp were corrected for nonspecific
binding by subtracting scans acquired in the presence of 100-fold
excess ATP. The peak fluorescence signal at 530 nm for TNP-ATP at each
concentration of ATP in the presence of Vi was determined, and the data
were normalized to the fluorescence of the sample with no ATP. The data
were fitted using GraphPad Prism 2.0 for the PowerPC Macintosh.
-32P]8-Azido-ADP from the
Pgp·8-Azido-ADP·Vi Complex Is Not Affected by Nucleotides--
The
results thus far show that following ATP hydrolysis, affinity for
nucleotide is considerably reduced. There is considerable evidence in
the literature that there is communication between the two ATP sites of
Pgp and that mutations or chemical modification of residues in either
ATP site precludes ATP hydrolysis (3, 6, 15, 20-25). This has led to
the hypothesis that there is alternating hydrolysis of ATP at the two
ATP sites (9, 26, 27), i.e. that nucleotide first binds to
one of the two sites but cannot be hydrolyzed. When another nucleotide
binds to the second ATP site it promotes hydrolysis at the first site,
which in turn powers substrate transport. In the context of our results this raises the following two questions. (a) Does nucleotide
binding per se affect Vi-induced trapping? (b)
Once ATP hydrolysis ensues, do nucleotides influence the release of ADP
from Pgp? To address the first question, we monitored Vi-induced
trapping of [
-32P]8-azido-ADP in the presence
increasing concentrations of AMPPNP, a nucleotide analogue, that can
bind to Pgp but cannot be hydrolyzed (10). Thus, if binding of
nucleotide at one ATP site exerts allosteric control over hydrolysis at
the other ATP site, binding of AMPPNP at one site should stimulate
Vi-induced trapping at the other site. Fig.
4A, however, clearly
demonstrates that AMPPNP inhibits in a dose- dependent
manner the Vi-induced trapping of [
-32P]8-azido-ADP
with an apparent Ki = 1.3 mM. This
suggests that binding of nucleotide at one site does not stimulate ATP hydrolysis at the other site.
View larger version (19K):
[in a new window]
Fig. 4.
Effect of nucleotides on the Vi-induced
trapping of [ -32P]8-azido-ADP
and on the dissociation of
[
-32P]8-azido-ADP from the
Pgp·[
-32P]8-azido-ADP·Vi
complex. A, effect of the nonhydrolyzable nucleotide
AMPPNP on Vi-induced trapping of
[
-32P]8-azido-ADP into Pgp. Crude membranes
(1 mg/ml protein) were incubated in the dark at 37 °C for 10 min
with 50 µM [
-32P]8-azido-ATP (3-5
µCi/nmol), 250 µM Vi, and increasing concentrations of
AMPPNP in the ATPase assay buffer. The reaction was stopped by adding
12.5 mM ice-cold ATP and placing the tubes on ice.
Following photo-cross-linking by UV irradiation at 365 nm for 5 min and
SDS-PAGE, the level of [
-32P]8-azido-ADP incorporated
was quantified using a PhosphorImager, and the data were fit by using
GraphPad Prism 2.0 for the PowerPC Macintosh. A single phase
exponential decay model best fits the data. B, nucleotides
have no effect on the rate of dissociation of
[
-32P]8-azido-ADP from the
Pgp·8-azido-ADP·Vi complex. Crude membranes (1 mg/ml protein) were
incubated in the dark at 37 °C for 10 min with 50 µM
[
-32P]8-azido-ATP (3-5 µCi/nmol) and 250 µM Vi in the ATPase assay buffer. The reaction was
stopped by adding 12.5 mM ice-cold ATP and placing the
tubes on ice. Untrapped nucleotides and excess Vi were removed by
centrifugation at 300,000 × g for 10 min, and the
membranes were resuspended in the Mg2+-free ATPase assay
buffer and divided into 4 aliquots. To these were added 10 mM MgCl2, control membranes; 10 mM
MgCl2 + 1.25 mM ATP; 10 mM
MgCl2 + 1.25 mM AMPPNP; or 10 mM
EDTA, respectively, and incubated at 37 °C. Aliquots were removed at
indicated intervals and placed on ice and photo-cross-linked by UV
irradiation at 365 nm for 5 min, and SDS-PAGE was performed on each
sample. The level of [
-32P]8-azido-ADP incorporated
was quantified using a PhosphorImager, and the data were fit using
GraphPad Prism 2.0 for the PowerPC Macintosh. Dissociation of
[
-32P]8-azido-ADP in control membranes (
) and in
the presence of 1.25 mM ATP (
), 1.25 mM
AMPPNP (
), or in Mg2+-free medium containing 10 mM EDTA (
) are shown in the figure.
-32P]8-azido-ADP from the
Pgp·8-azido-ADP·Vi complex. The [
-32P]8-azido-ADP
was trapped by incubating crude membranes with
[
-32P]8-azido-ATP and Vi at 37 °C for 10 min, and
free Vi and nucleotide were removed by centrifugation, and the
resuspended membranes were incubated at 37 °C under different
conditions. Aliquots were removed at intervals, and excess cold ATP was
added to displace the [
-32P]8-azido-ADP dissociated
from the complex, and the samples were cross-linked by UV irradiation.
Fig. 4B shows that the [
-32P]8-azido-ADP is
completely dissociated within 15 min at 37 °C with a
t1/2 of 2-3 min. The t1/2 for
dissociation is not influenced by the presence of excess 8-azido-ADP, 8-azido-ATP, ATP, or the nonhydrolyzable nucleotide AMPPNP, added in
the presence of Mg2+. The dissociation is, however,
strongly temperature-dependent with no appreciable dissociation
during a 15-min incubation at room temperature (data not shown). These
results suggest that following ATP hydrolysis, ADP dissociates
spontaneously and that while in the transition state, ADP or
8-azido-ADP is occluded and not exchangeable with exogenously added nucleotide.
-32P]8-azido-ADP trapping and release,
and the kinetic parameters for each step can be determined. In Fig.
5B we depict the rate constants for the repeating cycles of
Vi-induced [
-32P]8-azido-ADP trapping and release. The
data show that the rate constants for two hydrolysis events
(cf. Fig. 5B) are indistinguishable. The
occurrence of two ATP hydrolysis events in a single catalytic cycle
raises the question, is there a functional asymmetry between the two
ATP sites? For example, is the N-terminal ATP site committed to the
first hydrolysis event followed by the recruitment of the C-terminal
ATP site for the second ATP hydrolysis event? We addressed this
question by performing a mild trypsin digestion of Pgp after labeling
with [
-32P]8-azido-ADP at steps II, IV, and VI of the
catalytic cycle, as depicted in Fig. 5A. The results (Fig.
5C) clearly demonstrate that at high concentration of
[
-32P]8-azido-ATP, there is no preference for either
the N- or the C-terminal ATP site during any of the ATP hydrolysis
events.
View larger version (32K):
[in a new window]
Fig. 5.
Repeating cycles of ATP hydrolysis.
A, schematic representation of the experiment. Step
I and II, crude membranes (protein, 1 mg/ml) were
incubated in the dark at 37 °C with 50 µM
[ -32P]8-azido-ATP (3-5 µCi/nmol) and 250 µM Vi in the ATPase assay buffer. Aliquots were removed
at regular intervals over 15 min and placed on ice. The reaction was
stopped by adding excess ice-cold ATP (12.5 mM) and
photo-cross-linking by UV irradiation at 365 nm for 5 min on ice.
Steps II and III, at the end of 15 min, the
reaction mixture was transferred to ice, and un-trapped nucleotides and
excess Vi were removed by centrifugation at 300,000 × g for 10 min. The membranes were resuspended in the ATPase
assay buffer and placed at 37 °C. Aliquots were again removed at
indicated intervals over the next 20 min (35 min from start of
experiment), and photo-cross-linked as described above. Step
III and IV, at the end of 35 min, fresh 50 µM [
-32P]8-azido-ATP and 0.25 mM Vi were added to initiate the next cycle of ATP
hydrolysis. Aliquots were removed at regular intervals, over an
additional 15 min (50 min from start of experiment) and
photo-cross-linked as described above. At the end of 15 min the
reaction mixture was transferred to ice, and un-trapped nucleotides and
excess Vi were removed by centrifugation at 300,000 × g for 10 min, and the membranes were resuspended in the
ATPase assay buffer and placed at 37 °C. Steps IV and
V, the next cycle of dissociation of
[
-32P]8-azido-ADP was monitored by placing the
reaction mixture at 37 °C and removing aliquots as indicated above.
Steps V and VI, at the end of 20 min (70 min from
start of experiment) a third hydrolysis cycle was initiated by adding
50 µM [
-32P]8-azido-ATP and 0.25 mM Vi and followed over the next 10 min (80 min from start
of experiment). At the end of the experiment, all samples were
electrophoresed and the [
-32P]8-azido-ADP incorporated
into the Pgp band quantified using a PhosphorImager. The data were fit
using GraphPad Prism 2.0 for the Power Macintosh (see below). The
thick vertical arrows represent additions of
[
-32P]8-azido-ATP and Vi at steps I, III,
and V. B, the cycles of
[
-32P]8-azido-ADP trapping and release. The individual
fits are for the experiment described in A above. Initiation
of the first hydrolysis event (panels I and II),
dissociation of [
-32P]8-azido-ADP following the first
hydrolysis event (panels II and III), initiation
of the second hydrolysis event (panels III and
IV), dissociation of [
-32P]8-azido-ADP
following the second hydrolysis event (panels IV and
V), and initiation of the third hydrolysis event,
i.e. initiation of the next catalytic cycle (panels
V and VI). The data were fitted to a single phase
exponential decay model and the t1/2 was determined
for each step. C, distribution of Vi-trapped
[
-32P]8-azido-ADP in the two ATP sites. At steps
II, IV, and VI (described in Fig. 4A),
following photo-cross-linking, the samples were treated with trypsin
(protein:trypsin, 10:1) for 5 min at 37 °C to separate N- and C-terminal halves of Pgp (8). The reaction was stopped by
the addition of 5-fold excess soybean trypsin inhibitor, and SDS-PAGE
samples in the presence of 5 M urea were prepared by
incubation at 23 °C for 20 min. Following SDS-PAGE, radioactivity in
the N- and the C-terminal fragments in a dried gel were quantified
using a PhosphorImager. Figure shows distribution of
[
-32P]8-azido-ADP in the N- (filled bars)
and C-terminal (open bars) halves of Pgp at steps II,
IV, and VI as depicted in A above. The
values represent an average of 3 independent experiments, and the
standard deviations are shown by error bars.
-32P]8-azido-ADP incorporation and
release in a single catalytic cycle, and the filled bars
depict the extent of [125I]IAAP incorporated into Pgp at
various steps in the catalytic cycle (see Fig. 5, A and
B). The data for [
-32P]8-azido-ADP
incorporation and release, which have been quantitatively treated in
Fig. 5B, merely offer a visual representation in Fig. 6 of
the state of Pgp molecules in a single catalytic cycle.
View larger version (14K):
[in a new window]
Fig. 6.
Binding of [125I]IAAP to Pgp
during various steps of the catalytic cycle. Crude membranes (20 µg of protein) were labeled with 5 nM
[125I]IAAP at steps I-V in the catalytic
cycle of Pgp as depicted in Fig. 4A. The extent of
[125I]IAAP binding is superimposed on a representation of
Fig. 5B in a continuous time scale (shaded gray
area). The membranes were labeled with [125I]IAAP
after the following pretreatments, Step I, control untreated
membranes. Step II, membranes treated with 1.2 mM 8-azido-ATP and 250 µM Vi for 10 min at
37 °C. Step III, membranes at step II
centrifuged to remove excess 8-azido-ATP and Vi, resuspended in ATPase
buffer, and incubated for 15 min at 37 °C. Step IV,
membranes at the end of step III were again incubated with
1.2 mM 8-azido-ATP and 250 µM Vi for 10 min
at 37 °C. Step V, membranes in step IV were
centrifuged to remove excess 8-azido-ATP and Vi, resuspended in ATPase
buffer, and incubated for 15 min at 37 °C. Step Va
depicts an aliquot of membranes at step III treated with 1.2 mM 8-azido-ATP in the absence of Vi for 10 min at
37 °C.
-32P]8-azido-ATP in
the Presence of Vi--
To obtain the Km of
[
-32P]8-azido-ATP for Pgp during Vi-induced trapping,
crude membranes containing Pgp were incubated with increasing
concentrations of [
-32P]8-azido-ATP in the presence of
Vi at 37 °C. The reaction was stopped with excess ATP.
[
-32P]8-Azido-ADP incorporated into Pgp was quantified
from a SDS-PAGE gel as described under "Experimental Procedures."
The extent of [
-32P]8-azido-ADP trapping in the first
hydrolysis event is depicted in Fig.
7A. The single site model of
Henri-Michaelis-Menten best described the data to give a
Km of 18.4 µM. To determine the
kinetics of the second hydrolysis event, the crude membranes were first
allowed to trap 8-azido-ADP in the presence of Vi for 10 min; excess
8-azido-ATP and Vi were removed by centrifugation, and then the
occluded 8-azido-ADP was allowed to disassociate by incubation at
37 °C for 15 min (steps I and II in Fig.
5A). The second hydrolysis event was followed in the
presence of increasing concentrations of
[
-32P]8-azido-ATP and Vi, and the extent of
[
-32P]8-azido-ADP incorporation was quantified as
described above. The data (Fig. 7B) show that the
Km value for [
-32P]8-azido-ATP in
the presence of Vi during the second hydrolysis event, 19.4 µM, is indistinguishable from that for the first
hydrolysis event. However, the results depicted in Fig. 7 substantiate
the notion that although the hydrolysis events measured in Fig. 7, A and B, are kinetically similar, they occur in
two distinct conformations of Pgp vis à vis the
substrate binding (Figs. 5B and 6).
View larger version (18K):
[in a new window]
Fig. 7.
Determination of the
Km of
[ -32P]8-azido-ATP in the
presence of Vi-induced trapping during two ATP hydrolysis events in a
single catalytic cycle of Pgp. A, the
Km of [
-32P]8-azido-ATP during the
first hydrolysis event. Crude membranes were incubated with increasing
concentrations of [
-32P]8-azido-ATP (3-5 µCi/nmol)
in the presence of 250 µM Vi at 37 °C; at the end of
10 min the reaction was stopped with excess ATP.
[
-32P]8-Azido-ADP incorporated into Pgp was quantified
from a SDS-PAGE gel as described under "Experimental Procedures."
B, the Km of [
-32P]8-azido-ATP
during the second hydrolysis event. To study the kinetics of the second
hydrolysis event, the crude membranes were first allowed to trap
8-azido-ADP by incubating with 1.25 mM 8-azido-ATP in the
presence of 250 µM Vi for 10 min, and excess 8-azido-ATP
and Vi were removed by centrifugation, and then the occluded
8-azido-ADP was allowed to dissociate by incubation at 37 °C for 15 min. These membranes were then incubated with increasing concentrations
of [
-32P]8-azido-ATP in the presence of 250 µM Vi at 37 °C in the ATPase assay buffer. At the end
of 10 min, the reaction was stopped with excess ATP, and the
incorporation of radiolabel into Pgp was quantified as described above.
The data were analyzed using GraphPad Prism 2.0.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]8-azido-ADP through an entire
catalytic cycle, and we independently study the kinetics of the two
hydrolysis events. Crude membrane fractions as well as purified Pgp
reconstituted into lipid vesicles manifest a basal level of ATP
hydrolysis that is stimulated by substrates and modulators of Pgp by a
factor of up to 10 (3, 9). Pgp exhibits low affinity for ATP
(Km 0.3 to 1 mM) compared with, for
example, myosin or the mitochondrial F1F0-ATP
synthase (28, 29). Additionally, in Pgp no covalent phosphorylated (E-P) intermediate has been demonstrated as is known
to occur for the P-type ATPases (9). These facts led Senior and
co-workers (9, 26) to hypothesize that during ATP hydrolysis a state of
high chemical potential is generated and that the relaxation of such a
state powers the extrusion of substrate. Recent work from our
laboratory (10) experimentally demonstrated a large conformational
change accompanying ATP hydrolysis. We showed that the long lived
Pgp·8-azido-ADP·Vi transition state complex, which is generated
immediately following ATP hydrolysis, exhibits a drastic decrease in
the affinity for the substrate analogue [125I]IAAP. Even
more interesting was the observation that the release of the occluded
8-azido-ADP was not sufficient to regain substrate binding, which
occurred only after an additional ATP hydrolysis step. That two
hydrolysis events occur in a single catalytic cycle, one associated
with efflux of drug and the other to bring about conformational changes
that "reset" the molecule, raise several mechanistic questions
about ATP hydrolysis during the catalytic cycle of Pgp. Is there
conformational coupling between the drug-binding sites and the ATP
sites, and by extension what is the status of the drug and nucleotide
binding at each step in the catalytic cycle? What effect, if any, does
ATP hydrolysis have on subsequent nucleotide binding? Do the two ATP
hydrolysis events show different kinetics?
-32P]8-azido-ADP (Fig. 7A)
with the Ki (8-azido-ATP) for inhibition of
[125I]IAAP binding during trapping (Fig. 2A)
shows the two values to be almost identical (Km, 22 µM; Ki, 18 µM), and a
cause-effect relationship between the two is plausible. Moreover, the
fact that Vi-induced trapping of 8-azido-ADP inhibits substrate
([125I]IAAP) binding in a
concentration-dependent manner (Fig. 2A) provides stronger and more direct evidence for interactions between the
substrate- and nucleotide-binding sites. That these are long range
interactions resulting from conformational changes in the protein is
underscored by the fact that drugs do not affect nucleotide binding
per se (Fig. 1), nor do nucleotides affect substrate binding in the absence of ATP hydrolysis (10).
-32P]8-azido-ATP.
The Ki(8-azido-ATP) for inhibition of [
-32P]8-azido-ATP binding (12.3 µM) is
comparable to the
Km([
-32P]8-azido-ATP) for
Vi-induced trapping of [
-32P]8-azido-ADP (22 µM) and the Ki (8-azido-ATP) for
inhibition of [125I]IAAP binding (19 µM).
These data suggest that the conformational changes that follow ATP
hydrolysis reduce the affinity of both substrate and nucleotide for Pgp
and the extent of decrease in the affinity of the nucleotide;
[
-32P]8-azido-ATP (Figs. 2C and 3) is
comparable to that demonstrated earlier for the substrate
[125I]IAAP. The release of
[
-32P]8-azido-ADP from the complex is spontaneous and
not affected by nucleotide binding (Fig. 4A), and this is
sufficient for the next ATP hydrolysis event to ensue (Fig.
5B). The conformational changes occurring in the ATP sites
are thus different from those in the substrate site in that, except for
the release of occluded ADP, there is no additional energetic
requirement for resetting the nucleotide-binding pocket.
-32P]8-azido-ADP trapping and
[
-32P]8-azido-ADP release. Although the
t1/2 for each trapping and release event are
comparable, Fig. 6 shows how the conformational state of the molecule
differs during each of these events. It is clear from the
gray-shaded area in Fig. 6 (data shown in Fig.
5B) that the two hydrolysis events are indistinguishable. Concurrent measurements of substrate binding, however, distinguish different states of the Pgp molecule in which these two events occur.
Thus, when the first hydrolysis event is initiated (Fig. 6, step
I), [125I]IAAP binding is not affected in contrast
to over 90% inhibition of [125I]IAAP binding when the
second hydrolysis event is initiated (Fig. 6, step
III). At the end of the second hydrolysis event, however, [125I]IAAP binding is restored to normal levels (Fig. 6,
step V) bringing Pgp to a state comparable to step
I both in terms of substrate and nucleotide binding, suggesting
the completion of one catalytic cycle. It would thus be reasonable to
conclude that determining the kinetics of Vi-induced
[
-32P]8-azido-ADP trapping between steps I and II, as
depicted in Fig. 5, would represent the first hydrolysis event and
between steps III and IV the second event. Fig. 7 shows that the
Km ([
-32P]8-azido-ATP) for trapping
is identical for the two hydrolysis events. Thus, even though these
events have different functional consequences, they are kinetically
indistinguishable. This would suggest that it is unlikely that the two
hydrolysis events are individually associated with each ATP site. We
propose that the nucleotide-binding site for any hydrolysis event at
high nucleotide concentration is recruited randomly, that all
hydrolysis events are kinetically equivalent, and that the different
functional outcomes are a result of the conformational state of the Pgp
molecule when a particular hydrolysis event occurs. This view is also
consistent with the findings of Urbatsch et al. (31) that
the two ATP sites are functionally equivalent. The fact that following
Vi-induced trapping [
-32P]8-azido-ADP is distributed
equally in both N- and C-terminal ATP sites (Fig. 5C) at
steps II, IV, and VI, as depicted in Fig. 5A, further
supports this view.
-32P]8-azido-ADP labels the two ATP sites in equal
proportion (32, 33). Our results with recombinant human Pgp similarly
demonstrate that this distribution remains constant regardless of
whether it is monitored at step II, IV, or VI of the cycle as depicted in Fig. 5A (Fig. 5C). These results strongly
favor the conclusion that the two ATP sites are recruited randomly and
show similar kinetics, which emerge as a single
Km(ATP or 8-azido-ATP) during hydrolysis (6, 13, 15,
16). Moreover, the observation that trapping of 1 mol of ADP/mol of Pgp
is sufficient to block ATP hydrolysis (34, 35) has been interpreted to
mean that trapping of nucleotide at one site blocks catalysis at both
sites. This observation has in turn led to the speculation (26, 27) that the binding of nucleotide at one ATP site is not sufficient for
hydrolysis to occur and that the binding of nucleotide at the second
ATP site permits hydrolysis at the first site by an allosteric
mechanism. Data presented in this work, on the other hand, provide
direct experimental evidence that following ATP hydrolysis the affinity
of nucleotide for Pgp is drastically reduced (Figs. 2C and
3). Thus, the fact that trapping nucleotide at either ATP site blocks
hydrolysis at both can be explained by this conformational change that
drastically reduces the affinity of nucleotide for the second ATP site.
The reduced affinity for nucleotides to Pgp in the transition state is
also demonstrated by using the fluorescent nucleotide analogue, TNP-ATP
(Fig. 3, A and B). Such a perspective would also
be consistent with the characterization of ATP hydrolysis in Pgp (28)
which shows Henri-Michaelis-Menten kinetics with a single
Km in the 0.3-1 mM range, since no
cooperativity has been demonstrated vis à vis the
kinetics of ATP hydrolysis to suggest allosteric modulation. Finally,
as depicted in Fig. 4A, binding of the nonhydrolyzable
nucleotide AMPPNP does not stimulate Vi-induced trapping. If binding of
nucleotide at one ATP site exerts a positive allosteric control over
hydrolysis at the other, stimulation of hydrolysis should result in an
increase in Vi-induced nucleotide trapping.
View larger version (23K):
[in a new window]
Fig. 8.
A revised scheme for the catalytic cycle of
Pgp. The revised scheme is based on the data presented in this
paper as well as previous published work by us and others (8-10, 41).
The ellipses represent the substrate-binding sites, the "ON" and
the "OFF" site. The hexagon portrays the "ON" site with reduced
affinity for the drug. Two circles represent the ATP sites,
and the circles are shown overlapping to indicate that both
sites are required for ATP hydrolysis. The empty square with
rounded edges represents the ATP site with reduced affinity
for nucleotide. Step I, substrate binds to the "ON" site
of Pgp, and ATP binds to one or both of the two ATP sites. Step
II, ATP is hydrolyzed, and the drug is possibly moved to the lower
affinity "OFF" site. Step III, Pi is
released, and the drug extruded from Pgp at this step. Step
IIIA, when Pi is replaced by Vi, the Pgp·ADP·Vi
complex is generated, which exhibits a reduced affinity for both
substrate as well as nucleotide. Step IV, the ADP and Vi
dissociate from the complex. Although the ATP sites have reverted to
the "high affinity" state, the affinity for drug substrate
continues to be low. Step V, following dissociation of the
ADP in step IV, an additional molecule(s) of ATP is
hydrolyzed. Step VI, Pi is released. Step
VIA, when Pi is replaced by Vi, the Pgp·ADP·Vi
complex is generated, which continues to exhibit a reduced affinity for
substrate. Steps VIB/VII, the dissociation of ADP allows the
conformation of Pgp to be restored to its original state with high
affinity for substrate binding. The initiation of the next catalytic
cycle follows. Although we depict ATP binding and hydrolysis as
occurring in ATP site in the N-half of Pgp, our data do not suggest any
preference in the presence of saturating concentrations of ATP or
8-azido-ATP, for either of the two sites, and the process appears to be
random (see Fig. 5C). The observed drastically reduced
affinity of nucleotides for the second ATP site, while the first one is
in the catalytic conformation (steps II and V),
provides a basis for the proposed alternate catalytic cycle model (9).
In addition, our recent work (16) indicates that ADP release at
steps IV and VII (underlined) appears
to be the rate-limiting steps in the catalytic cycle of ATP
hydrolysis.
The model we propose invokes two hydrolysis events (Fig. 8, steps
II and V) during each catalytic cycle. We have also
demonstrated here that the two hydrolysis events in a single catalytic
cycle are kinetically identical (Figs. 5B and 7) and differ
only with respect to the status of the molecule vis à
vis substrate binding (Fig. 6). This too is consistent with the
notion that ATP does not show preferential affinity to either ATP site,
and the sites are recruited randomly for hydrolysis (see above). The
recent resolution of the crystal structure of the soluble ATP subunit, ArsA of the bacterial arsenite efflux pump (37) and ATP subunits of
bacterial ABC transporters MutS (38, 39) and MalK (40) that exhibit
structural and functional similarity to Pgp, shows that the two
functional ATP sites, are each composed of residues from both the N-
and the C-terminal ATP sites. Such a tertiary structural organization
is plausible for Pgp where the nucleotide binding domains in the N- and
C-terminal halves of the protein each contribute to both ATP binding
and hydrolysis with similar kinetic properties. The resolution of the
structure of the Pgp ATP domains and further work on site-directed
mutagenesis of residues in ATP sites should provide additional insights
into the mechanism of the catalytic cycle of ATP hydrolysis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael M. Gottesman for discussions and encouragement and Dr. Marianna Müller for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Cell
Biology, Division of Basic Sciences, NCI, Bldg. 37, Rm. 1B-22, National
Institutes of Health, 37 Convent Dr., Bethesda, MD 20892-4255. Tel.:
301-402-4178; Fax: 301-435-8188; E-mail: ambudkar@helix.nih.gov.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M011294200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Pgp, P-glycoprotein; ABC, ATP-binding cassette; AMPPNP, 5'-adenylylimididiphosphate; [125I]IAAP, [125I]io- doarylazidoprazosin; PAGE, polyacrylamide gel electrophoresis; Vi, vanadate; TNP-ATP, 2'-O-(trinitrophenyl)adenosine 5'-triphosphate; MDR, multidrug resistance; MES, 2-[N-morpholino]ethane sulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gottesman, M. M., Pastan, I., and Ambudkar, S. V. (1996) Curr. Opin. Genet. & Dev. 6, 610-617[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ambudkar, S. V., Dey, S., Hrycyna, C. A., Ramachandra, M., Pastan, I., and Gottesman, M. M. (1999) Annu. Rev. Pharmacol. Toxicol. 39, 361-398[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Sarkadi, B.,
Price, E. M.,
Boucher, R. C.,
Germann, U. A.,
and Scarborough, G. A.
(1992)
J. Biol. Chem.
267,
4854-4858 |
5. | Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8472-8476[Abstract] |
6. | Urbatsch, I. L., al-Shawi, M. K., and Senior, A. E. (1994) Biochemistry 33, 7069-7076[Medline] [Order article via Infotrieve] |
7. | Ford, J. M., and Hait, W. N. (1990) Pharmacol. Rev. 42, 155-199[Medline] [Order article via Infotrieve] |
8. |
Dey, S.,
Ramachandra, M.,
Pastan, I.,
Gottesman, M. M.,
and Ambudkar, S. V.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10594-10599 |
9. | Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Sauna, Z. E.,
and Ambudkar, S. V.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2515-2520 |
11. |
Ambudkar, S. V.,
Cardarelli, C. O.,
Pashinsky, I.,
and Stein, W. D.
(1997)
J. Biol. Chem.
272,
21160-21166 |
12. | Shapiro, A. B., and Ling, V. (1998) Eur. J. Biochem. 254, 189-193[Abstract] |
13. | Ramachandra, M., Ambudkar, S. V., Chen, D., Hrycyna, C. A., Dey, S., Gottesman, M. M., and Pastan, I. (1998) Biochemistry 37, 5010-5019[CrossRef][Medline] [Order article via Infotrieve] |
14. | Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve] |
15. |
al-Shawi, M. K.,
Urbatsch, I. L.,
and Senior, A. E.
(1994)
J. Biol. Chem.
269,
8986-8992 |
16. |
Kerr, K. M.,
Sauna, Z. E.,
and Ambudkar, S. V.
(2001)
J. Biol. Chem.
276,
8657-8664 |
17. | Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647[Medline] [Order article via Infotrieve] |
18. |
Lerner-Marmarosh, N.,
Gimi, K.,
Urbatsch, I. L.,
Gros, P.,
and Senior, A. E.
(1999)
J. Biol. Chem.
274,
34711-34718 |
19. | Liu, R., and Sharom, F. J. (1997) Biochemistry 36, 2836-2843[CrossRef][Medline] [Order article via Infotrieve] |
20. | Hrycyna, C. A., Ramachandra, M., Germann, U. A., Cheng, P. W., Pastan, I., and Gottesman, M. M. (1999) Biochemistry 38, 13887-13899[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Loo, T. W.,
and Clarke, D. M.
(1995)
J. Biol. Chem.
270,
22957-22961 |
22. |
Muller, M.,
Bakos, E.,
Welker, E.,
Varadi, A.,
Germann, U. A.,
Gottesman, M. M.,
Morse, B. S.,
Roninson, I. B.,
and Sarkadi, B.
(1996)
J. Biol. Chem.
271,
1877-1883 |
23. |
Loo, T. W.,
and Clarke, D. M.
(1995)
J. Biol. Chem.
270,
21449-21452 |
24. | Senior, A. E., and Bhagat, S. (1998) Biochemistry 37, 831-836[CrossRef][Medline] [Order article via Infotrieve] |
25. |
al-Shawi, M. K.,
and Senior, A. E.
(1993)
J. Biol. Chem.
268,
4197-4206 |
26. | Senior, A. E., and Gadsby, D. C. (1997) Semin. Cancer Biol. 8, 143-150[CrossRef][Medline] [Order article via Infotrieve] |
27. | Senior, A. E. (1998) Acta Physiol. Scand. Suppl. 643, 213-218[CrossRef][Medline] [Order article via Infotrieve] |
28. | Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) J. Bioenerg. Biomembr. 27, 31-36[Medline] [Order article via Infotrieve] |
29. | Sharom, F. J., Yu, X., Chu, J. W., and Doige, C. A. (1995) Biochem. J. 308, 381-390[Medline] [Order article via Infotrieve] |
30. | Scarborough, G. A. (1995) J. Bioenerg. Biomembr. 27, 37-41[Medline] [Order article via Infotrieve] |
31. | Urbatsch, I. L., Gimi, K., Wilke-Mounts, S., and Senior, A. E. (2000) Biochemistry 39, 11921-11927[CrossRef][Medline] [Order article via Infotrieve] |
32. | Sankaran, B., Bhagat, S., and Senior, A. E. (1997) Biochemistry 36, 6847-6853[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Urbatsch, I. L.,
Sankaran, B.,
Bhagat, S.,
and Senior, A. E.
(1995)
J. Biol. Chem.
270,
26956-26961 |
34. |
Urbatsch, I. L.,
Sankaran, B.,
Weber, J.,
and Senior, A. E.
(1995)
J. Biol. Chem.
270,
19383-19390 |
35. |
Urbatsch, I. L.,
Gimi, K.,
Wilke-Mounts, S.,
and Senior, A. E.
(2000)
J. Biol. Chem.
275,
25031-25038 |
36. | Liu, R., and Sharom, F. J. (1996) Biochemistry 35, 11865-11873[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Zhou, T.,
Radaev, S.,
Rosen, B. P.,
and Gatti, D. L.
(2000)
EMBO J.
19,
4838-4845 |
38. | Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710[CrossRef][Medline] [Order article via Infotrieve] |
39. | Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Diederichs, K.,
Diez, J.,
Greller, G.,
Muller, C.,
Breed, J.,
Schnell, C.,
Vonrhein, C.,
Boos, W.,
and Welte, W.
(2000)
EMBO J.
19,
5951-5961 |
41. | Urbatsch, I. L., Beaudet, L., Carrier, I., and Gros, P. (1998) Biochemistry 37, 4592-4602[CrossRef][Medline] [Order article via Infotrieve] |