From the S. C. Johnson Medical Research Center, Mayo Foundation, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
Received for publication, October 13, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Multidrug resistance-associated protein
(MRP1) transports solutes in an ATP-dependent manner by
utilizing its two nonequivalent nucleotide binding domains (NBDs) to
bind and hydrolyze ATP. We found that ATP binding to the first NBD of
MRP1 increases binding and trapping of ADP at the second domain (Hou,
Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as
indicating that the binding of ATP at NBD1 causes a conformational
change in the molecule and increases the affinity for ATP at NBD2.
However, we did not distinguish between the possibilities that the
enhancement of ADP trapping might be caused by either ATP binding alone
or hydrolysis. We now report the following. 1) ATP has a much lesser
effect at 0 °C than at 37 °C. 2) After hexokinase treatment, the
nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate),
does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue,
adenosine 5'-( Multidrug resistance is a major obstacle to successful
chemotherapeutic treatment of many types of cancers. Over-expression of
P-glycoprotein (P-gp)1 and/or
multidrug resistance-associated protein (MRP1) confers resistance to a
broad range of anti-cancer drugs (1, 2). Both proteins transport
anticancer drugs out of cells in an ATP-dependent manner by
utilizing their membrane-spanning domains and two nucleotide binding
domains (NBDs) (3-5), i.e. they couple ATP binding and hydrolysis to transport of solutes (6-15). However, it is unknown whether they share the same mechanism of this coupling. In the extensively studied P-gp, the two NBDs have been shown to be
functionally equivalent with identical ATP hydrolysis steps occurring
alternately at each NBD (16-20) and coupling one transport event with
one ATP hydrolysis (21). Ambudkar's group (22) reported that there are
two independent ATP hydrolysis events in a single drug transport cycle,
one ATP hydrolysis is associated with efflux of drug, whereas the other
causes conformational resetting to the original state of the molecule
(23). However, in their interpretation the ATP binding/hydrolysis sites of P-gp are recruited in a random manner during hydrolysis (23), meaning that the two NBD sites are functionally equivalent. In other reports, the two NBDs of P-gp were found to be
essential for its function but not entirely symmetric (24, 25). Vigano
et al. (25) proposed recently that ATP binding/hydrolysis at
NBD1 is associated with efflux of drug, whereas the event at NBD2 is
associated with the "reset" of the molecule. Therefore, how events
at NBD1 and NBD2 of P-gp cooperate during drug transport is still not
clear. Considerable evidence has accumulated indicating that the two
NBDs of some other ATP-binding cassette (ABC) transporters, including the sulfonylurea receptor (SUR1) (26, 27), cystic fibrosis transmembrane-conductance regulator (CFTR) (28, 29), and MRP1
(30-33), have very distinctive properties. For example, in the case of
MRP1, the following points clearly indicate that its two nonequivalent
NBDs have different properties and functions. First, modifications of
the consensus Walker motifs in the two NBDs do not inactivate the
protein completely and have different effects on solute transport
(30-32). Second, photoaffinity labeling experiments with 8-azido-ATP
also revealed an asymmetry between NBD1 and NBD2, with NBD1
preferentially labeled by 8-N3[ Materials--
Sodium orthovanadate, EGTA, ATP, ATP Cell Line Expressing Wild-type MRP1 in BHK Cells and Cell
Culture--
Stable cell line expressing wild-type MRP1 in baby
hamster kidney (BHK-21) cells was established previously (31, 35). These cells were cultured in Dulbecco's modified Eagle's medium/F-12 with 5% fetal bovine serum supplemented with 150 µM
methotrexate (the original colonies expressing MRP1 were selected in
500 µM methotrexate and decreased to 150 µM
methotrexate because the lesser amount of this drug did not affect the
expression of MRP1 protein) at 37 °C in 5% CO2. Cells
for membrane vesicle preparations were grown in roller bottles (Bellco)
in Dulbecco's modified Eagle's medium/F-12 media containing 5% fetal
bovine serum and 150 µM methotrexate at 37 °C.
Membrane Vesicle Preparations--
MRP1-containing membrane
vesicles were prepared according to the procedure described previously
(31). Briefly, the cells grown in roller bottles were collected by
centrifugation, resuspended in membrane vesicle preparation buffer
containing 10 mM Tris-HCl, pH 7.5, 250 mM
sucrose, 0.2 mM MgCl2 and 1× protease
inhibitors (2 µg/ml aprotinin, 121 µg/ml benzamidine, 3.5 µg/ml
E64, 1 µg/ml leupeptin, and 50 µg/ml Pefabloc) and equilibrated on
ice for 20 min at 800 p.s.i. in a Parr N2 cavitation
bomb. After pressure release, the cell homogenate was adjusted to 1 mM EDTA. The homogenate was diluted 5-fold with 10 mM Tris-HCl, pH 7.5, and 25 mM sucrose and
centrifuged at 1000 × g to remove nuclei and unbroken
cells. The supernatant was overlaid on a 35% sucrose solution
containing 10 mM Tris-HCl, pH 7.5, and 1 mM
EDTA and centrifuged at 16,000 × g for 30 min.
Membranes at the interface were collected, diluted 5-fold with a
solution containing 10 mM Tris-HCl, pH 7.5, and 250 mM sucrose, and then centrifuged at 100,000 × g for 45 min. The pellet was resuspended in a solution
containing 10 mM Tris-HCl, pH 7.5, 250 mM
sucrose, and 1× protease inhibitors. After passage through a
LiposofastTM vesicle extruder (200-nm filter, Avestin, Ottawa, Canada),
the membrane vesicles were aliquoted and stored in
Hexokinase Treatment of ATP Analogues--
To remove the trace
amount of contaminating ATP in ATP analogues, 5 mM AMP-PNP,
AMP-PCP, or ATP Photoaffinity Labeling of MRP1 Protein--
Vanadate preparation
and photoaffinity labeling of MRP1 protein were performed according to
procedures described previously (31). Briefly, the photolabeling
experiments were carried out in a 10-µl solution containing 10 µg
of membrane vesicles from MRP1 expressing cells, 10 µM
8-N3[ Purification of MRP1 Protein--
The previous procedure (35)
used to purify MRP1 protein from BHK cells was modified slightly. Cells
collected from roller bottles were washed with a solution containing 10 mM Tris-HCl, pH 7.5, 250 mM sucrose, and 0.2 mM MgCl2, and the cell pellet was kept in a
[ ATP Enhancement of ADP Trapping in MRP1 Protein Is Greatly
Diminished on Ice--
We found that photolabeling of MRP1 protein
with 8-N3[ Hexokinase-treated AMP-PNP Does Not Enhance ADP Trapping by
MRP1--
The nonhydrolyzable ATP analogue, AMP-PNP, enhanced ADP
trapping by MRP1 protein (34), supporting the hypothesis that
nucleotide binding alone can cause the conformational change of the
protein. However, this could be misleading if there is a trace amount
of hydrolyzable nucleotide contaminant. This can be removed efficiently by treating the solution with hexokinase (36). The results in Fig.
2 show that AMP-PNP, after hexokinase
treatment, did not enhance ADP trapping by MRP1 protein, but instead it
inhibited ADP trapping. Previous results clearly indicated that
8-N3[ The Nonhydrolyzable ATP Analogue, AMP-PCP, Enhances ADP
Trapping--
If nucleotide binding alone cannot cause the
conformational change leading to increased trapping, then the binding
of other nonhydrolyzable ATP analogues, such as AMP-PCP, should also
not enhance ADP trapping. To test this hypothesis, both
hexokinase-treated and untreated AMP-PCP were utilized in the ADP
trapping experiments. Fig. 3,
A and B, shows that AMP-PCP, which was not
treated with hexokinase, enhanced ADP trapping ~50%, whereas the
hexokinase-treated AMP-PCP did so by ~20% (Fig. 3, C and
D). The enhancing effect of the untreated AMP-PCP is
slightly greater than the treated compound, presumably reflecting the
removal of a trace amount of ATP. These results may mean that
nucleotide binding at NBD1 alone can induce the conformational change
required to enhance ADP trapping at NBD2. However, this interpretation
is not consistent with the conclusion derived from Fig. 2. Therefore we
postulated that in some nucleotides, such as ATP or AMP-PCP,
binding alone without hydrolysis can induce this conformational change,
whereas in other nucleotides, such as AMP-PNP, binding cannot induce
this change.
ATP ATP
Because it takes ~20 min for one MRP1 molecule to hydrolyze one
ATP Over-expression of MRP1 protein confers resistance to a broad
range of anti-cancer drugs (2). Solutes (for example, anti-cancer drugs) are extruded out of cells by MRP1 protein in an
ATP-dependent manner (7, 14, 37) by utilizing its two NBDs
to bind and hydrolyze ATP. Both NBDs of MRP1 protein can bind
nucleotides (30, 31, 33). However, the properties and the functions of
the two NBDs do not seem to be equal as discussed in the Introduction. How are the events at NBD1 and NBD2 related during solute transport? We
have found that the binding of ATP at NBD1 enhances ADP trapping or
AMP-PNP binding to NBD2, implying that ATP binding at NBD1 causes
conformational change of the MRP1 molecule (34) and the trapping of
ADP·Vi, mimicking the ATP hydrolysis intermediate ADP·Pi, enhances
intact ATP binding at NBD1 (31), implying that ATP hydrolysis at NBD2
causes conformational change in the MRP1 molecule. These conformational
changes may contribute to active solute transport by the molecule.
However, it is not clear whether the enhancing effect of nucleotide
binding at NBD2 requires ATP hydrolysis at NBD1. The nonhydrolyzable
ATP analogue, AMP-PNP, slightly enhances ADP trapping to MRP1 protein
(34), implying that nucleotide binding alone can induce the
conformational change. However, why was the enhancing effect of AMP-PNP
much lower than that of ATP (34)? Interestingly, a poorly hydrolyzable
ATP analogue, ATP How does ATP binding to NBD1 induce the conformational change? The
original "unexcited" structure of MRP1 protein should be the same
no matter whether ATP, ATP Although these data provide evidence that ATP binding, not hydrolysis,
at NBD1 is sufficient to induce the conformational change and enhance
nucleotide binding at NBD2, they do not speak directly to how the
protein couples ATP hydrolysis to solute transport. Upon binding of ATP
to NBD2 there should be a transient state in which both NBDs bound ATP.
Whether the bindings of ATP to both NBD1 and NBD2 will lead to the
formation of a transient ATP sandwich (41-43) between NBD1 and NBD2 is
not known. However, the efficient hydrolysis of the bound ATP at NBD2
(30, 31) may cause conformational change of the MRP1 protein (34) and
generate the negatively charged products, ADP and phosphate. The
electrostatic repelling force between them may facilitate the release
of the hydrolysis products from NBD2. What is the fate of the ATP bound
at NBD1 during or after release of the ATP hydrolysis products,
phosphate and ADP, from NBD2? When the dually expressed N- and C-halves were labeled with 8-N3[,
-methylene)triphosphate, whether
hexokinase-treated or not, causes a slight enhancement. 4) In
contrast, the hexokinase-treated poorly hydrolyzable ATP analogue,
adenosine 5'-O-(thiotriphosphate) (ATP
S), enhances ADP
trapping to a similar extent as ATP under conditions in which ATP
S
should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not
required to enhance ADP trapping by MRP1 protein; 2) with nucleotides
having appropriate structure such as ATP or ATP
S, binding alone can
enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping
is greatly diminished when the MRP1 protein is in a "frozen state"
(0 °C); and 4) the steric structure of the nucleotide
-phosphate is crucial in determining whether binding of the
nucleotide to NBD1 of MRP1 protein can induce the conformational change
that influences nucleotide trapping at NBD2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(30, 31), whereas NBD2 trapped the nucleoside diphosphate hydrolysis
product (30, 31, 33). Third, the ATP binding/hydrolysis sites of MRP1
seem not to be recruited in a random manner because photolabeling by
the nonhydrolyzable 8-N3[
-32P]AMP-PNP
occurred predominately at NBD1 (34), and the NBD1 fragment was labeled
predominantly with 8-N3[
-32P]ATP on ice in
the dual-expressed N- and C-halves of MRP1 (30). Fourth, ADP trapping
at NBD2 enhances ATP binding at NBD1 (31), and ATP binding at NBD1
allosterically enhances ADP trapping or AMP-PNP binding at NBD2 (34),
implying that ATP binding at NBD1 or ADP trapping at NBD2 induces
conformational change of the MRP1 molecule. It seems likely that the
conformational changes of the MRP1 molecule caused by ATP
binding/hydrolysis provide mechanical force to pump drug out of the
cell. However, experiments to date have not directly determined whether
ATP binding alone or hydrolysis at NBD1 can cause conformational change
of the MRP1 protein and enhance ADP trapping at NBD2. We have now done
this and found that ATP hydrolysis is not required to enhance ADP
trapping, and ATP binding alone is sufficient to enhance ADP trapping.
In addition, the steric position of the
-phosphate of the nucleotide
is a crucial factor in determining whether or not the binding of the nucleotide to NBD1 of MRP1 protein can enhance ADP trapping.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S,
AMP-PNP, AMP-PCP, LiCl, ouabain, and sheep brain lipid were purchased
from Sigma. Formic acid and polyethyleneimine-cellulose plates were
from Fisher. His-Bind Resin was from Novagen.
N-Dodecyl-
-D-maltoside (DDM) was from
Calbiochem. 8-N3[
-32P]ADP was purchased
from Affinity Labeling Technologies. [
-35S]ATP
S was
purchased from Amersham Biosciences. Dulbecco's modified Eagle's
medium/F-12 cell culture media were from Invitrogen. Hexokinase was from Roche Molecular Biochemicals. The Stratalinker UV Crosslinker 2400 model (wavelength 254 nm) was from Stratagene.
80 °C.
S solutions were treated with hexokinase as described
previously (36). Briefly, each nucleotide at 5 mM in
a 1-ml solution containing 20 mM Tris-HCl, pH 8.0, 10 mM glucose, 10 mM MgCl2, and 20 units of hexokinase was incubated at 30 °C for 30 min. Hexokinase in
the solution was removed by passing the solution through a Centricon 10 microconcentrator (Amicon, molecular weight cut-off 10,000).
-32P]ADP (1.2 µCi), 40 mM Tris-HCl, pH 7.5, 2 mM ouabain, 800 µM vanadate, 10 mM MgCl2, and 0.1 mM EGTA. The amounts of other nucleotides added, incubation
time, and temperature are indicated specifically in the figure legends.
The samples were then transferred to ice and diluted with 500 µl of
ice-cold Tris-EGTA buffer (40 mM Tris-HCl and 0.1 mM EGTA, pH 7.5). The membranes were pelleted in a
microcentrifuge in a cold room (4 °C), resuspended in 10 µl of
Tris-EGTA buffer, placed on ice, and irradiated for 2 min in a
Stratalinker UV Crosslinker (
= 254 nm). The labeled proteins
were separated by polyacrylamide gel (7%) electrophoresis and
electroblotted to a nitrocellulose membrane.
80 °C freezer overnight. The cell pellet was resuspended in a
solution containing 10 mM Hepes, pH 7.2, 1 mM
EDTA, and 1× protease inhibitors, and then the cells were transferred
to a Dounce homogenizer. After seven strokes in this homogenizer, the same volume of a solution containing 10 mM Hepes, pH 7.2, 1 mM EDTA, and 500 mM sucrose was added to the
homogenizer, and then another six strokes were performed. Nuclei were
removed by centrifugation at 300 × g for 15 min at
4 °C. Membranes were collected at 33,000 × g for 45 min at 4 °C. The membrane pellet was resuspended in a binding buffer
containing 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 20% glycerol, 25 mM imidazole, 1% DDM, 0.4% sheep brain
lipid, and 0.05%
-mercaptoethanol and sonicated for a short time on ice. The insoluble material was removed by centrifugation at
10,000 × g for 15 min. The supernatant was applied
onto a His-Bind Resin column that had been pre-equilibrated with the
binding buffer. The column was washed with 6 column volumes of modified
binding buffer containing 0.1% DDM and 25 mM imidazole
(first wash), 6 column volumes of modified binding buffer containing
0.1% DDM and 40 mM imidazole (second wash), and 6 column
volumes of modified binding buffer containing 20 mM
Tris-HCl, pH7.4, 0.1% DDM and 40 mM imidazole (third
wash). The bound protein was eluted with 2 column volumes of buffer
containing 20 mM Tris-HCl, pH 7.4, 500 mM NaCl,
20% glycerol, 300 mM imidazole, 0.1% DDM, 0.4% sheep brain lipid, and 0.05%
-mercaptoethanol. The eluate was dialyzed against a solution containing 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 20% glycerol, 0.1% DDM, and 0.05%
-mercaptoethanol.
-35S]ATP
S Hydrolysis--
The experiments
were performed in a 15-µl solution containing 40 mM
Tris-HCl, pH 7.5, 2 mM ouabain, 0.1 mM EGTA, 10 mM MgCl2, 100 µM
[
-35S] ATP
S (3 µCi), and 0.5 µg of purified
MRP1 protein. The same amount of protein-free sheep brain lipid (26 µg) solution as in the 0.5 µg of purified MRP1 protein was used as
a negative control. The reaction mixture was brought back to
ice, and 10% aliquots were spotted immediately on a
polyethyleneimine-cellulose plate. The samples were chromatographed
with 0.5 M LiCl and 1 M formic acid as the
solvent. The amounts of intact [
-35S]ATP
S and
[35S]phosphate in each reaction mixture were determined
by electronic autoradiography using a Packard Instant Imager (Packard
Instrument Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ADP was enhanced 4-fold
mainly at NBD2 (34), suggesting that ATP binding or hydrolysis at NBD1
caused conformational change of the protein and increased affinity for
ATP at NBD2. However, these experiments were performed at 37 °C and
did not distinguish whether ATP binding or hydrolysis caused the
conformational change of the protein. To distinguish these two
possibilities, the same photolabeling experiments were performed on
ice. Fig. 1, A and B, showed that the trapping of ADP to the protein was
enhanced ~30-40% in the presence of 5 to 20 µM ATP.
The trapping was inhibited almost 70% in the presence of 640 µM ATP, simply because of competition between ATP binding
and ADP trapping. These results were interpreted in the following two
ways: 1) ATP hydrolysis at NBD1 may be required to induce the
conformational change of the molecule to enhance ADP trapping at NBD2
and limited ATP hydrolysis on ice greatly diminishes the enhancement
effect; 2) ATP binding alone can cause a conformational change that
increases ADP trapping at NBD2 at higher temperature, such as 37 °C,
and the smaller augmentation of trapping by ATP on ice may reflect a
membrane structure and/or MRP1 protein that are in a "frozen
state." Therefore these results cannot be used to distinguish the two
possibilities mentioned above.
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Fig. 1.
ATP-dependent enhancement effect
on
8-N3[ -32P]ADP
trapping is greatly diminished on ice. The photolabeling
experiments were performed according to the procedures described under
"Experimental Procedures" in a 10-µl reaction mixture
containing varying concentrations of ATP, indicated above
the gel, on ice for 10 min. A, autoradiogram of ADP trapping
on MRP1 protein in the presence of varying concentrations of ATP. The
molecular weight markers are indicated on the left. The
arrow indicates the
8-N3[
-32P]ADP-labeled 190-kDa MRP1
protein. The 45-kDa protein labeled by
8-N3[
-32P]ADP was also present in the
membrane vesicles prepared from the parental BHK cells and was not
recognized by several different antibodies against MRP1 protein.
B, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein versus ATP concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel A were determined by electronic
autoradiography (Packard Instant Imager) and plotted out against ATP
concentrations. The amount of
8-N3[
-32P]ADP incorporated into MRP1
protein in the absence of ATP was considered as 100%. The results are
the average of three independent experiments.
-32P]AMP-PNP bound to NBD1 of MRP1
(34). Therefore it seems likely that nucleotide binding alone, at least
in the case of AMP-PNP, cannot enhance ADP trapping by MRP1.
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Fig. 2.
Hexokinase-treated AMP-PNP does not enhance
8-N3[ -32P]ADP
trapping to MRP1 protein. The photolabeling experiments were
performed according to the procedures described under "Experimental
Procedures" in a 10-µl reaction mixture containing varying
concentrations, indicated above the gel, of
hexokinase-treated AMP-PNP at 37 °C for 10 min. Hexokinase treatment
of AMP-PNP solution is described under "Experimental Procedures."
A, autoradiogram of ADP trapping to MRP1 protein in the
presence of varying concentrations of AMP-PNP. The molecular weight
markers are indicated on the left. The arrow
indicates the 8-N3[
-32P]ADP-labeled
190-kDa MRP1 protein. B, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein versus AMP-PNP concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel A were determined by electronic
autoradiography and plotted out against AMP-PNP concentrations. The
amount of 8-N3[
-32P]ADP incorporated into
MRP1 protein in the absence of AMP-PNP was considered as 100%. The
results are the average of five independent experiments.
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Fig. 3.
AMP-PCP slightly enhances
8-N3[ -32P]ADP
trapping to MRP1 protein. The photolabeling experiments were
performed according to the procedures described under "Experimental
Procedures" in a 10-µl reaction mixture containing varying
concentrations, indicated above the gel, of either untreated
(A) or hexokinase-treated (C) AMP-PCP at 37 °C
for 10 min. Hexokinase treatment of AMP-PCP solution is described under
"Experimental Procedures." A, autoradiogram of ADP
trapping to MRP1 protein in the presence of varying concentrations of
AMP-PCP (untreated). The molecular weight markers are indicated on the
left. The arrow indicates the
8-N3[
-32P]ADP-labeled 190-kDa MRP1
protein. B, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein versus AMP-PCP concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel A were determined by electronic
autoradiography and plotted out against AMP-PCP concentrations. The
amount of 8-N3[
-32P]ADP incorporated into
MRP1 protein in the absence of AMP-PCP was considered as 100%. The
results are the average of three independent experiments. C,
autoradiogram of ADP trapping on MRP1 protein in the presence of
varying concentrations of hexokinase-treated AMP-PCP. D,
plot of the amount of 8-N3[
-32P]ADP
incorporated into MRP1 protein versus AMP-PCP
concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel C were determined by electronic
autoradiography and plotted out against hexokinase-treated AMP-PCP
concentrations. The amount of
8-N3[
-32P]ADP incorporated into MRP1
protein in the absence of AMP-PCP was considered as 100%. The results
are the average of five independent experiments.
S Enhances ADP Trapping--
To further test the above
hypothesis, ATP
S was utilized in the ADP trapping experiments. Fig.
4, A and B, show
that ATP
S enhanced ADP trapping to MRP1 protein almost 3-fold.
Similarly, hexokinase-treated ATP
S also increased the trapping
~3-fold (Fig. 4, C and D). Because the
enhancing effect of ATP
S is much greater than that of AMP-PCP (Fig.
3) and slightly less than that of ATP (34), ATP
S may have an effect
on ADP trapping similar to that of ATP under other conditions, such as
at 0 °C. The experiments in Fig. 4E were performed on ice
and showed that ATP
S stimulated ADP trapping by MRP1 protein
~20-30% (Fig. 4F). These results imply that ATP
S
binding alone can cause the underlying conformational change.
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Fig. 4.
ATP S enhances
8-N3[
-32P]ADP
trapping to MRP1 protein. The photolabeling experiments were
performed according to the procedures described under "Experimental
Procedures" in a 10-µl reaction mixture containing varying
concentrations, indicated above the gel, of either untreated
(A) or hexokinase-treated (C) ATP
S at 37 °C
for 10 min. Hexokinase treatment of ATP
S solution is described under
"Experimental Procedures." A, autoradiogram of ADP
trapping on MRP1 protein in the presence of varying concentrations of
ATP
S (not hexokinase-treated). The ATP
S concentration in each
reaction mixture is indicated above the gel. The molecular
weight markers are indicated on the left. The
arrow indicates the
8-N3[
-32P]ADP-labeled 190-kDa MRP1
protein. B, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein versus ATP
S concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel A were determined by electronic
autoradiography and plotted out against ATP
S concentrations. The
amount of 8-N3[
-32P]ADP incorporated into
MRP1 protein in the absence of ATP
S was considered as 100%. The
results are the average of two independent experiments. C,
autoradiogram of ADP trapping to MRP1 protein in the presence of
varying concentrations of hexokinase-treated ATP
S. The ATP
S
(hexokinase-treated) concentration in each reaction mixture is
indicated above the gel. D, plot of the amount of
[
- 8-N3[
-32P]ADP incorporated into
MRP1 protein versus ATP
S concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel C were determined and plotted out against
hexokinase-treated ATP
S concentrations. The amount of
8-N3[
-32P]ADP incorporated into MRP1
protein in the absence of ATP
S was considered as 100%. The results
are the average of three independent experiments. E,
autoradiogram of ADP trapping on MRP1 protein in the presence of
varying concentrations of hexokinase-treated ATP
S at 0 °C for 10 min. F, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein versus ATP
S concentrations. The amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein in panel E were determined and plotted out against
hexokinase-treated ATP
S concentrations. The results are the average
of four independent experiments. Because the enhancement effects are
always calculated by comparing the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein in the absence of ATP
S, the amounts of
8-N3[
-32P]ADP incorporated into MRP1
protein at 0 and 37 °C are important factors. On the basis of 19 different experiments performed at 0 and 37 °C at the same time, the
labeling at 37 °C in the absence of other nucleotide is 1.95 ± 0.67-fold higher than at 0 °C. The value in the control lane of
C was 4044, whereas it was 2060 in the control lane of
E (4044/2060 = 1.96).
S Binding without Hydrolysis Can Enhance ADP
Trapping--
Thus far, we have not ruled out the possibility that the
poorly hydrolyzable ATP analogue, ATP
S, might be hydrolyzed to some extent during the 10-min incubation period at 37 °C. Therefore we
estimated how long it would take to hydrolyze one ATP
S molecule. Table I shows that it took ~20 min for
one MRP1 molecule at 37 °C to hydrolyze one ATP
S. Therefore,
~50% of the ATP
S bound to MRP1 protein should be hydrolyzed
during the 10-min of incubation at 37 °C. Hence, some hydrolysis did
occur.
Time required to hydrolyze one [-35S]ATP
S by one MRP1
molecule
S, at shorter times hydrolysis is essentially negligible. The
experiments in Fig. 5A were
performed as follows. The pelleted membrane proteins containing MRP1
were resuspended on ice with a 10-µl ice-cold reaction mixture,
transferred to a 37 °C water bath, and incubated for 1, 2, 4, 8, 16, or 32 min. The samples were brought back to ice after incubation
for the indicated times at 37 °C and diluted with 500 µl of
ice-cold Tris-EGTA buffer immediately, and then the membrane was
pelleted by centrifugation in a cold room (4 °C). Therefore, the
temperature inside of the tubes incubated at 37 °C for only 1 min
should not be 37 °C at the beginning of the incubation, and the
incubation time at 37 °C must be less than 1 min. The amount of
ATP
S hydrolyzed during this short period must be much less than
during the 32-min period (Fig. 5A, lane
32'). Yet, this sample (1-min incubation at 37 °C) had the
greatest enhancing effect on ADP trapping (Fig. 5, A and
C), indicating that ATP
S hydrolysis was not responsible for the conformation change and that ATP
S binding alone was
sufficient. Fig. 5C shows that the enhancing effects
gradually decreased with incubation time. The mechanism of this
diminution is not yet clear. However, one of the possible reasons is
that the unstimulated ADP trapping in the absence of ATP
S gradually
increased with incubation time (Fig. 5, A and B),
implying that occlusion or trapping of ADP by vanadate takes time or
requires conformational change induced by ATP binding at NBD1. ATP
S
did not significantly enhance ADP trapping if the samples were
incubated on ice (Fig. 5A, lanes 0',
and
+). This result is consistent with the data in Figs. 1 and
4E showing that no matter if ATP or ATP
S was employed, the nucleotides did not significantly enhance the ADP trapping if the
experiments were performed on ice.
View larger version (28K):
[in a new window]
Fig. 5.
ATP S hydrolysis is
not required for enhancing
8-N3[
-32P]ADP
trapping to MRP1 protein. The photolabeling experiments were
performed according to the procedures described under "Experimental
Procedures" in a 10-µl reaction mixture containing either no
ATP
S (
lanes) or 20 µM
hexokinase-treated ATP
S (+ lanes). The samples were
resuspended in a 10-µl ice-cold reaction mixture on ice, transferred
immediately to a 37 °C water bath, and then incubated at 37 °C
for the indicated time. Lanes 0',
and +, the samples were
incubated on ice for 1 min without transferring to 37 °C water bath.
The other samples were incubated at 37 °C for 1 min (1',
and +),
2 min (2',
and +), 4 min (4',
and +), 8 min (8',
and +), 16 min (16',
and +), and 32 min (32',
and +). The samples were
washed with 500 µl of ice-cold Tris-EGTA buffer immediately after the
37 °C incubation and UV irradiated on ice for 2 min. Because the
samples were transferred from 0 to 37 °C directly, the temperature
inside of the tubes was not 37 °C at the beginning of the 37 °C
incubation. A, autoradiogram of ADP trapping to MRP1 protein
in the absence or presence of 20 µM hexokinase-treated
ATP
S. The
or + signs above the gel indicate the
condition of in the absence or presence of 20 µM of
hexokinase-treated ATP
S in each of the specific reaction. The
molecular weight markers are indicated on the left. The
arrow indicates the
8-N3[
-32P]ADP-labeled 190-kDa MRP1
protein. B, plot of the amount of
8-N3[
-32P]ADP incorporated into MRP1
protein. The amounts of 8-N3[
-32P]ADP
incorporated into MRP1 protein in panel A were determined by
electronic autoradiography. C, enhancement effect of ATP
S
on ADP trapping. The amount of
8-N3[
-32P]ADP incorporated into MRP1
protein in the absence of ATP
S was considered as 100%. The results
are the average of four independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, induced the conformational change to the same
extent as ATP as determined by fluorescence quenching (38), implying
that nucleotide binding alone may be responsible for induction of the conformational change. However, because ATP
S is a poorly
hydrolyzable ATP analogue, the nucleotide used in the experiments might
be hydrolyzed by MRP1. Therefore, the question of whether ATP binding or hydrolysis is required to induce the conformational change was still
unanswered. Our present results clearly indicate that ATP binding alone
is sufficient to induce the conformational change of MRP1 protein. The
results in Fig. 2 show that hexokinase-treated AMP-PNP cannot enhance
ADP trapping, implying that AMP-PNP binding to NBD1 (34) cannot induce
the necessary conformational change. If nucleotide binding alone cannot
induce the conformational change, then the binding of other
nonhydrolyzable ATP analogues should also not induce the conformational
change. However, in contrast to AMP-PNP, another nonhydrolyzable ATP
analogue, AMP-PCP, can enhance ADP trapping (Fig. 3), implying that
nucleotide binding alone can induce the conformational change of the
protein. Interestingly, the enhancing effect of AMP-PCP on ADP trapping
was much less than that of ATP, perhaps because of the structural
difference between AMP-PCP and ATP. If the structure difference between
nucleotides is a major factor determining the enhancing effects, then a
nucleotide with a structure similar to that of ATP, such as ATP
S,
should have a similar effect. Indeed, the hexokinase-treated ATP
S
had an effect similar to that of ATP (Fig. 4), although we had not ruled out the possibility that ATP
S might be hydrolyzed during the
10-min incubation at 37 °C. The results shown in Table I and Fig. 5
clearly indicate that ATP
S binding alone, not hydrolysis, can
enhance ADP trapping in MRP1 protein. However, nucleotide binding on
ice (30) greatly diminished the stimulatory effect (Figs. 1 and 4),
implying that ATP binding alone can induce the conformational change
only under proper conditions such as those of temperature. In
conclusion: 1) nucleotide hydrolysis at NBD1 is not required to induce
the conformational change stimulating ADP trapping at NBD2; 2)
nucleotide binding alone under proper conditions is sufficient to
induce the conformational change; 3) the proper steric structure of the
-phosphate of the nucleotide is a crucial factor affecting the
ability of the nucleotide to induce the conformational change; when
nitrogen replaces oxygen between the
and
phosphates in
AMP-PNP, this eliminates the enhancing effect, whereas when carbon
replaces oxygen in AMP-PCP this greatly reduces the enhancing effect;
4) the replacement of oxygen with sulfur on the
-phosphate in
ATP
S also slightly reduces the enhancing effect.
S, AMP-PNP, or AMP-PCP has been utilized
to excite MRP1 protein. The only difference between ATP, AMP-PNP, and
AMP-PCP is the atom between the
- and
-phosphates, which
determines the distance and angle between the
- and
-phosphates (39). Therefore the steric structure of the
-phosphate is the crucial determinant of the structural perturbation. Which residues of
MRP1 protein interact with the
-phosphate when the nucleotide binds
to NBD1? Although the three-dimensional structure of MRP1 protein has
not been solved, the structures of other ATP-binding cassette
transporters, such as periplasmic histidine permease of
Salmonella typhimurium (40) and MJ0796 from
Methanococcus jannaschii (41), may provide a clue. By
analogy with these, Ser-685 (a residue in Walker A of NBD1),
Gln-713 (
-phosphate linker), and Asp-792 (a residue in Walker B of
NBD1) may interact with the
-phosphate via Mg2+. Lys-684
(a residue in Walker A of NBD1) may interact with the
-phosphate
directly. Asp-793 may function as a catalytic base to attack the water
molecule to hydrolyze the bound ATP. Val-680 and Gly-681 (residues in
the Walker A motif of NBD1) and Ser-1431, Val-1432, and Gly-1433
(residues in the ATP-binding cassette signature sequence of
NBD2) may interact with the
-phosphate. Upon ATP binding to NBD1 the
-phosphate of the bound nucleotide either "pushes" or
"pulls" (by electrostatic interactions between the
-phosphate
and the charged residues) some of these residues, leading to the
conformational change and increasing the affinity for ATP at NBD2 (34).
Changing of the atom between the
- and
-phosphates changes the
spatial orientation of the
-phosphate, either eliminating (such as
AMP-PNP) or diminishing (such as AMP-PCP) the pushing or pulling
force. If that is the case, mutations of these residues may affect ATP
binding at NBD1 and decrease the ATP enhancing effect on ADP trapping.
Indeed, mutations of K684L and D792A greatly diminish the ATP enhancing
effect on ADP trapping (34). Interestingly, mutation of D792E, which
did not significantly change the negative charge at that position, also
greatly diminished the ATP enhancement effect on ADP
trapping,2 indicating that
the distance between the negative charged residue, Asp-792, and the
-phosphate of ATP is also very important.
-32P]ATP on ice,
the NBD1 fragment was predominantly labeled (30). However, when the
experiments were performed at 37 °C, NBD2 was predominantly labeled
(30). Consistent with the above finding, when full-length
wild-type MRP1 was labeled with
8-N3[
-32P]ATP on ice and digested with
trypsin, the labeling at NBD1 was greater than at NBD2.2 In
contrast, when the experiments were performed at 37 °C, NBD2 was
predominantly labeled (31). We interpreted these results to mean that
the ATP bound at NBD1 was released during the incubation period at
37 °C. If that is the case, whether the ATP bound at NBD1 is
released as an intact ATP or hydrolyzed first and then released is not
known. No matter how the ATP bound at NBD1 is released, the releasing
of this bound nucleotide may bring the MRP1 molecule back to the
original unexcited state so that the MRP1 molecule can start another
cycle of solute transport.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sharon Fleck for preparation of the manuscript, Marv Ruona for preparation of the graphics, and Dr. Andrei Aleksandrov for discussion of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant CA89078 from NCI, National Institutes of Health.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: S. C. Johnson Medical
Research Center, Mayo Clinic Scottsdale, 13400 East Shea Blvd.,
Scottsdale, AZ 85259. Tel.: 480-301-6206; Fax: 480-301-7017; E-mail:
xbchang@mayo.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210480200
2 Y.-x. Hou, J. R. Riordan, and X.-b. Chang, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
P-gp, P-glycoprotein;
MRP1, multidrug resistance-associated protein;
NBD, nucleotide binding domain;
8-N3ADP, 8-azidoadenosine
5'-diphosphate;
AMP-PNP, adenyl 5'-(yl iminodiphosphate);
AMP-PCP, adenosine 5'-(,
-methylene)triphosphate;
ATP
S, adenosine
5'-O-(thiotriphosphate);
DDM, N-dodecyl-
-D-maltoside;
BHK, baby hamster
kidney.
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