From the Structure and Function of Biological
Membranes-Center of Structural Biology and Bioinformatics, Free
University of Brussels, B-1050 Brussels, Belgium and the
¶ S. C. Johnson Medical Research Center, Mayo Clinic Arizona,
Scottsdale, Arizona 85259
Received for publication, August 5, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and
transports chemotherapeutic drugs as well as diverse organic anions
such as leukotriene LTC4. The transport of
chemotherapeutic drugs requires the presence of reduced GSH. By
using hydrogen/deuterium exchange kinetics and limited trypsin
digestion, the structural changes associated with each step of the drug
transport process are analyzed. Purified MRP1 is reconstituted into
lipid vesicles with an inside-out orientation, exposing its
cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both
ATP-dependent drug transport and drug-stimulated ATPase
activity. Our results show that during GSH-dependent drug
transport, MRP1 does not undergo secondary structure changes but only
modifications in its accessibility toward the external environment.
Drug binding induces a restructuring of MRP1 membrane-embedded domains
that does not affect the cytosolic domains, including the nucleotide
binding domains, responsible for ATP hydrolysis. This demonstrates that
drug binding to MRP1 is not sufficient to propagate an allosteric
signal between the membrane and the cytosolic domains. On the other
hand, GSH binding induces a conformational change that affects the
structural organization of the cytosolic domains and enhances ATP
binding and/or hydrolysis suggesting that GSH-mediated conformational
changes are required for the coupling between drug transport and ATP
hydrolysis. Following ATP binding, the protein adopts a conformation
characterized by a decreased stability and/or an increased
accessibility toward the aqueous medium. No additional change in
the accessibility toward the solvent and/or the stability of this
specific conformational state and no change of the transmembrane
helices orientation are observed upon ATP hydrolysis. Binding of a
non-transported drug affects the dynamic changes occurring during ATP
binding and hydrolysis and restricts the movement of the drug and its release.
Multidrug resistance-associated protein 1 (MRP1),1 a member of the
ATP-binding cassette transporter family, confers resistance to a wide
range of chemotherapeutic drugs including anthracyclines, vinca alkaloids, and epipodophyllotoxins by exporting them
out of cells (1-6). ATP binding and hydrolysis as well as the presence of GSH is essential for transport to occur (2, 3, 7-14). In addition,
MRP1 transports anionic compounds such as LTC4 (8, 15),
bilirubin glucuronide (16), glucuronide, and sulfate-conjugated estrogens (17, 18) and bile salts (19), glutathione disulfide (20), and
arsenical and antimonial oxyanions (2).
Its predicted topology is characteristic of a typical ABC transporter:
a core structure containing two membrane-spanning domains (MSD1 and
MSD2), each of them composed of six transmembrane (TM) segments and two
nucleotide-binding domains (NBD1 and NBD2) that are located at the
cytoplasmic face of the membrane (6, 21-23). In addition to this core
structure, MRP1 contains a third NH2-terminal membrane-spanning domain (MSD0) made of five transmembrane segments (21, 24). The role of this domain in the function of MRP1 remains to be
clarified (25).
Drug binding to MRP1 occurs through interactions with the transmembrane
domains (26-32), whereas the NBDs are responsible for ATP
binding/hydrolysis and for coupling energy release to substrate transport. Recently, low resolution images of MRP1 have been obtained from single particle image analysis and electron microscopy of two-dimensional crystals (33). The projection structures give the first
static image of the tertiary organization of MRP1. Nevertheless, no
information about the structural modifications induced upon GSH and
drug binding at different steps of MRP1-mediated drug transport process
is available.
To gain further insight into the mechanism by which MRP1-mediated drug
transport occurs, we have characterized the intermediate structural
states associated with each step of the drug transport, and we observed
the influence of GSH on these conformational changes. Purified MRP1 was
reconstituted into lipid vesicles with an inside-out orientation,
exposing its cytoplasmic region to the external medium (34). The
resulting proteoliposomes have been shown previously to exhibit both
ATP-dependent drug transport and drug-stimulated ATPase
activity (34).
We have utilized limited proteolytic cleavage and infrared spectroscopy
to identify and characterize distinct intermediate structural states.
The data suggest that GSH induces structural modifications that allow
the coupling between drug transport and ATP hydrolysis. Binding of ATP,
rather than hydrolysis, increases the accessibility of MRP1 to the
aqueous medium. Such a conformational change may be required for the
release of the drug in the external medium. No additional change in the
accessibility toward the solvent and no change in the orientation of
the transmembrane helices are observed upon ATP hydrolysis. Therefore
we suggest that ATP hydrolysis could change the affinity of the
substrate through conformational changes involving rotation of the
transmembrane helices along their axis.
Binding of a non-transported drug reorganizes the membrane-embedded
domains in a conformation that restricts the movement of the drug and
its release.
Materials--
ATP, AMP-PNP, GSH, doxorubicin (DOX), and
N- Purification and Reconstitution of MRP1--
Stable baby hamster
kidney (BHK-21) cell line expressing MRP1 was generated by using
procedures described previously (35). The conditions of expression and
purification were as described (35). Reconstitution of the purified
MRP1 was achieved using SM-2 Bio-Beads to remove detergent from
protein/detergent/lipid mixture as described previously (34).
Attenuated Total Reflection-Fourier Transform Infrared
Spectroscopy (ATR-FTIR)--
ATR-FTIR spectra were recorded, at room
temperature, on a Bruker IFS55 FTIR spectrophotometer equipped with a
liquid nitrogen-cooled mercury-cadmium-telluride detector at a nominal
resolution of 2 cm Sample Preparation--
The sample contained 20 µg of
reconstituted MRP1. For the measurements carried out in the presence of
different ligands, the molar protein to ligand ratio is 1:14 for DOX
and FCE, 1:3 for GSH, and 1:6 for ATP or AMP-PNP. Thin films of
oriented multilayers were obtained by slowly evaporating the sample on
one side of the ATR plate under a stream of nitrogen (36). The ATR
plate was then sealed in a universal sample holder.
Secondary Structure Analysis--
The sample on the ATR plate
was rehydrated by flushing D2O-satured N2 for
2 h at room temperature. 512 scans were averaged for each
measurement. The determination of the secondary structure was based on
the shape of the amide I band (1600-1700 cm Orientation of the Secondary Structures--
The orientation of
different secondary structures was determined as described previously
(42, 43). Spectra were recorded with the incident light polarized
parallel and perpendicular with respect to the incidence plane.
Dichroism spectra were computed by subtracting the perpendicular
polarized spectrum from the parallel polarized spectrum. The
subtraction coefficient was chosen so that the area of the lipid ester
band at 1740 cm
An upward deviation on the dichroism spectrum indicates a dipole
oriented preferentially near a normal to the ATR plate. Conversely, a
downward deviation on the dichroism spectrum indicates a dipole oriented closer to the plane of the ATR plate (36). The dichroic ratio
RATR is defined as the ratio of the amide I area recorded
for the parallel polarization (A H/D Exchange Kinetics--
20 µg of reconstituted
MRP1 were deposited on a germanium plate as described above. Nitrogen
was saturated with D2O by bubbling in a series of three
vials containing D2O. Before starting the deuteration, 10 spectra of the sample were recorded to test the stability of the
measurements. At zero time, the D2O-saturated N2 flux, at a flow rate of 100 ml/min, was connected to the
sample. For each kinetic time point, 24 scans were recorded and
averaged at a resolution of 2 cm Measurements of ATPase Activity--
ATPase activity of
reconstituted MRP1 was measured by following the release of the
inorganic phosphate using a colorimetric method (48). Proteoliposomes
(4 µg of MRP1) were incubated in 10 mM Hepes, pH 7.4, 6 mM MgCl2, 3 mM ATP, in the absence
or in the presence of 20 µM DOX or FCE and 3 mM GSH, over a 2-h period at 37 °C. The contribution of
MgATP, drugs, or GSH to the colorimetric assay was subtracted.
Limited Proteolysis by Trypsin--
Reconstituted MRP1, in 10 mM Hepes, pH 7.4, was preincubated with different ligands
(3 mM GSH, 20 µM anthracycline and 3 mM ATP or AMP-PNP) for 10 min at 37 °C before trypsin
digestion was performed. Trypsin was added to the mixture (trypsin/MRP1
ratio is 1:400, w/w) and incubation carried out at 25 °C. After the desired times of incubation, aliquots were removed from the mixture, and the reaction was stopped by adding a trypsin inhibitor solution (TLCK) to 50 µg/ml. Zero time controls were obtained by adding trypsin inhibitor before adding trypsin. The peptide fragments were
separated through a Tris-Tricine polyacrylamide gel (16.5% T, 3% C)
and visualized by silver staining.
Effects of different ligands on trypsin activity were assayed using
BAPNA as substrate. The tryptic activity was tested at room temperature
in 200 µl of medium containing 10 mM Hepes, pH 7.4, 10 µg of trypsin, 0.5 mM BAPNA with or without ligands. The enzymatic activity of trypsin was determined by measuring the absorbance of p-nitroanilide at 405 mM. This
absorbance was recorded immediately after mixing and continued every
minute for 10 min and every 2 min until 30 min, on a Labsystems iEMS
Reader MF spectrophotometer.
The aim of the present study was to investigate how GSH affects
the structure of MRP1 at different steps of the transport cycle,
i.e. the drug-binding step and the ATP-binding and
hydrolysis step. Experiments are conducted in the presence of two
anthracycline derivatives that either do accumulate or do not
accumulate in resistant cells. Doxorubicin, a widely clinically used
anticancer agent, is actively extruded from cells overexpressing MRP1
(2-4). In contrast, a doxorubicin analogue,
3'-(3'-methoxymorpholino-doxorubicin (FCE), accumulates identically in
drug-sensitive and -resistant cells overexpressing MRP1, demonstrating
that this compound is not transported by MRP1 (49). Conformational
changes induced by binding and hydrolysis of ATP are analyzed in the
presence of MgAMP-PNP and MgATP. The use of MgAMP-PNP, a
nonhydrolyzable analog of MgATP, allows discrimination between the
influence of nucleotide binding and nucleotide hydrolysis on MRP1 structure.
The study is performed on purified MRP1 reconstituted into liposomes of
asolectin as described previously (34). The reconstituted MRP1 retains
its ATPase and transport activity. The orientation of MRP1 into
liposomes was investigated by comparison of its ATPase activity before
and after permeabilization of liposomes in the presence of 2 mM Chaps. This Chaps concentration has been shown previously to permeabilize liposomes (34, 50). We observed that the
ATPase activity was not enhanced by addition of 2 mM Chaps
indicating that MRP1 is inserted into liposomes in an inside-out configuration (34).
Secondary Structure Analysis--
The infrared spectrum of MRP1
alone, in the 1800 to 1400 cm
The secondary structure of MRP1 in the absence or in the presence of
ligands was determined by Fourier deconvolution and curve-fitting analysis of the amide I band of a deuterated sample as described previously (39). The proportions of different secondary structures are
shown in Table I and indicate that drug
and GSH binding in the absence or in the presence of ATP or AMP-PNP do
not modify MRP1 secondary structure.
Orientation in MRP1--
Spectra of MRP1 are recorded with
parallel and perpendicular polarized light (Fig.
2, A and B).
Dichroic spectra are obtained by subtracting the perpendicular from the
parallel polarized spectrum (Fig. 2C). The orientation of
the acyl chains of lipids is assessed using characteristic bands
associated with lipid molecules. Specific spectral bands, such as
It is the dichroism of the amide I band that characterizes the
orientation of MRP1 transmembrane domains. The dichroic spectrum (Fig.
2C) displays, in all cases, a positive dichroism in the amide I region with a maximum at 1654 cm
The mean orientation of the transmembrane helices obtained from the
dichroic ratios is evaluated around 42° with respect to the normal to
the germanium plate, considering an orientation of the amide I dipole
of 27° (42, 44, 51). Such a tilt has been found for the transmembrane
segments in crystal of an ABC transporter namely MsbA, a lipid flippase
from Escherichia coli (52). The mean orientation of the
helices is not significantly modified by the presence of different
ligands (Table II). Simultaneous binding
of FCE, GSH, and MgATP modifies the mean orientation of the
helices.
Kinetics of Deuteration--
At constant experimental conditions
(pH and temperature), the rate of hydrogen/deuterium exchange is
related to the solvent accessibility to the NH amide groups of the
protein, which in turn is related to the tertiary structure of the
protein and to the stability of a specific conformational state as well
as the secondary structure of the protein. Because the IR analysis of the secondary structure of MRP1 in the presence of different ligands shows that its secondary structure is not modified, differences in H/D
exchange rate will reflect changes in the tertiary structure of the
protein and/or in the stability of a specific conformational state.
Amide hydrogen exchange was followed by monitoring the amide II
absorption peak (maximum at 1544 cm
MgATP binding and hydrolysis generate distinct conformations depending
on the nature of the drug bound to the protein. DOX and GSH are
simultaneously bound to the protein, and addition of MgATP increases
the accessibility of the protein toward the solvent and/or decreases
the stability of the conformational state as shown by a decrease of the
unexchanged hydrogens proportion from ~40 to 32% (Fig. 4).
Replacement of MgATP, by its nonhydrolyzable analog, MgAMP-PNP, has no
effect on the H/D exchange rate observed with MgATP (Fig. 4).
In contrast, when FCE and GSH were simultaneously bound, addition of
MgATP led to an increase of the percentage of unexchanged hydrogens
from 40 to 53% (Fig. 4). As observed for DOX, this decrease of the
accessibility of the protein to the aqueous phase and/or increase of
the stability of the conformational state is due to the ATP binding,
rather than ATP hydrolysis because in the presence of MgATP or a
nonhydrolyzable analog, MgAMP-PNP, the kinetics are similar (Fig.
4).
Because the H/D exchange is a first-order reaction, the exchange curve
is expected to display a multiexponential decay corresponding to the
different groups of amide protons characterized by a common period
Ti (Equation 3),
The large number of protons makes it impossible to obtain the
individual rate constants. One approach to this problem is to choose
arbitrarily a number of exponentials to fit the exchange curves. We
have chosen three exponentials of amide groups, characterized by their
period Ti (i = 1-3) and their
proportion ai. In order to compare the proportion
ai of each amide group for all the experimental
curves, the same Ti values were used for all the
fittings. These periods were chosen as follows: T1 = 1 min for the very fast exchanging protons
(a1), T2 = 9 min for
the intermediate (a2), and
T3 = 666 min for the very slow exchanging
protons (a3) (Table
III). In the absence of any ligand, ~33% of the amide hydrogens was exchanged for deuterium within 1 min, and an additional 28% after 9 min. The remaining 39% hydrogens exchanged much slower and represent the very inaccessible regions of
MRP1. In the presence of GSH, a large fraction of the very fast
exchanging protons became inaccessible to the aqueous medium (about
21% or 321 amino acids). Drug binding has the opposite effect. Very
slow exchanging protons are now very quickly exchanged with deuterium
(about 11% or 168 amino acids). The simultaneous presence of GSH and
drugs results in an MRP1 conformation for which exchange kinetics is
similar to that of the protein alone. In the presence of ATP or its
nonhydrolyzable analog AMP-PNP, the kinetics is quite dependent on the
nature of the drug. When doxorubicin is bound, subsequent ATP binding
exposes the protein to its aqueous environment, and the slow exchanging
population of protons decreases with a concomitant increase of the
fraction of protons exchanging at a fast rate (Table III). When ATP is
hydrolyzed, the slow exchanging population of protons decreases with a
concomitant increase of the fraction of protons exchanging at
intermediate rate (Table III). The situation is quite different with
FCE, a non-transported drug. When ATP binds and is hydrolyzed, the fast exchanging protons behave essentially as very slow exchanging protons
(Table III).
Limited Proteolysis of MRP1 with or without Ligands--
Trypsin
cleavage site accessibility has been successfully used to investigate
ligand-induced conformational changes of various proteins, including
ABC transporters (P-glycoprotein, PMP70) (53-55) and ATPases
(K+,H+-ATPase) (56). In our experiments,
reconstituted MRP1 was incubated (10 min) with different substrates
before being subjected to trypsin digestion. At various times, aliquots
were removed from the mixture and added to the trypsin inhibitor
solution. The trypsin digests were analyzed on Tris-Tricine gels.
The effect of ligands on trypsin activity is assayed using BAPNA as
substrate (Table IV). The trypsin to
protein ratio is adjusted in order to maintain the same trypsin
activity under various circumstances. Therefore, changes in the trypsin
digestion profile cannot be attributed to differences in trypsin
activity but reflect a different susceptibility of MRP1 to tryptic
cleavage upon ligand binding.
Fig. 5 (lanes
2-6) shows that incubation of MRP1 with trypsin resulted in a
gradual disappearance of the full-length ~190-kDa MRP1 and
concomitant appearance of peptide fragments with approximate molecular
masses of 130, 115, 75, and 60 kDa. These fragments have been
identified previously using antibodies raised against defined epitopes
corresponding to different regions of MRP1 (21, 24, 28, 29, 57). The
115 and 75-kDa fragments correspond to the cleavage at the
trypsin-sensitive site in the cytoplasmic region linking NBD1 to
MSD2 (L1) (Fig. 6). The 115-kDa fragment encodes sequences that include MSD0, L0, MSD1, and NBD1; the 75-kDa fragment corresponds to the COOH-terminal fragment containing MSD2 and
NBD2. The exact location of the second trypsin cleavage site generating
the 130- and 60-kDa fragments remains contradictory and is assigned
either in the cytoplasmic loop (L0) linking MSD0 to MSD1 or in the
cytoplasmic loop CL4 (Fig. 6). Although identification of the exact
cleavage site responsible for the generation of the 130 and 60 kDa is
complicated by the attachment of N-linked oligosaccharide chains, the 130-kDa band must contain NBD1 and NBD2 (24, 57). Therefore, the 60-kDa fragment represents either MSD0 and part of L0
cytoplasmic loop or MSD0, L0 cytoplasmic loop, and the two first
transmembrane helices of MSD2. These fragments are subsequently digested by prolonged trypsin incubation.
GSH has a marked protective effect against digestion of MRP1 by trypsin
(Fig. 5, lanes 7-11). This is consistent with a
conformational change affecting the two sensitive trypsin sites that
are less exposed to the solvent. GSH binding increases also the
stability of fragments at 130, 115, 75, and 60 kDa suggesting that
further degradation of these fragments in the connecting loops is also prevented. In contrast, the presence of drugs (DOX or FCE) (Fig. 7, lanes 7-16) does not
modify the pattern of digestion obtained for the protein alone (Fig.
7, lanes 2-6) indicating that the protein is just as
sensitive to trypsin in the drug bound-state as the protein alone. In
the presence of both GSH and DOX (Fig. 8,
lanes 7-11) or GSH and FCE (Fig. 8, lanes
12-16), the whole protein is less efficiently digested compared
with the protein alone (Fig. 8, lanes 2-6). The digestion
profile is very similar to that obtained with GSH alone (Fig. 5,
lanes 7-11) confirming that only GSH affects the structure
of the cytosolic domains.
In the presence of MgAMP-PNP, a nonhydrolyzable analog of MgATP, DOX,
and GSH (Fig. 9, lanes
12-16), the pattern of digestion is drastically modified compared
with the one of protein alone (Fig. 9, lanes 2-6). The
sensitivity to trypsinolysis for the full-length protein and the 130-, 115-, 75-, and 60-kDa fragments is increased, as judged by their rapid
and total disappearance on the gel. This increased exposure facilitates
the attack of trypsin on the proteolytic site(s) located in these
regions. The same digestion profile is observed when DOX (Fig. 9,
lanes 12-16) is replaced with FCE (Fig. 9, lanes
7-11). When MgAMP-PNP is replaced with MgATP, a similar enhanced
sensitivity to the trypsin is observed (Fig.
10), demonstrating that MgATP binding,
rather than MgATP hydrolysis, induces a conformational change that
makes the cytoplasmic loops more accessible to the solvent.
ATPase Activity Measurements--
The ATPase activity of
reconstituted MRP1 is about 8 nmol/mg/min. FCE or DOX binding does not
modify significantly the rate of ATP hydrolysis (Fig.
11), whereas 21% stimulation is
observed in the presence of 3 mM GSH. No additional
stimulatory effect is detected when drugs and GSH are bound
simultaneously to MRP1. Hooijberg et al. (58) have reported
a similar stimulation of the ATPase activity by GSH in MRP1-containing
membrane vesicles; transported drugs vincristine and daunorubicin do
not activate the enzyme.
GSH is required for MRP1 to confer cell resistance to
chemotherapeutic drugs including anthracyclines, vinca
alkaloids, and etoposide (7-14). How GSH facilitates the transport of
these drugs is not yet known, but it is clearly not related to the
sulfhydryl reducing capacity of this tripeptide because
S-methyl GSH and other short chain alkyl derivatives of GSH
also facilitate transport (9, 18). The proposal that these anticancer
drugs are pumped out of cells by MRP1 via a co-transport mechanism with
free GSH is still a matter of debate (6, 9, 14). Structural information about the effect of GSH binding to MRP1 at different stages of the
transport cycle is certainly required for understanding this complex
mechanism. As this information is not available, we decided to
investigate the conformational changes induced upon GSH interaction with the protein in the absence or in the presence of amphiphilic drugs
(i.e. anthracyclines) and ATP, in order to characterize structural intermediate states involved in the MRP1-mediated transport process.
We combined different experimental approaches to gain structural
information about the cytosolic and membrane-embedded domains of MRP1.
First, quantitative global information about the conformational changes
occurring in the protein was obtained by monitoring the amide hydrogen
exchange. Second, IR-polarized attenuated total reflection was used to
gain information about the orientation of MRP1 transmembrane domains.
Third, trypsin digestion and ATPase activity assays allowed us to
investigate structural changes of the cytosolic domains.
Several conclusions can be drawn from this study. First, MRP1 does not
undergo detectable secondary structure changes upon interaction with
its substrates (Table I).
Does ligand binding affect MRP1 tertiary structure? A change in the
tertiary structure is supported by the H/D exchange rate measurements
demonstrating that after drug (DOX and FCE) binding, the accessibility
toward the solvent is increased and/or the stability of the
conformational state is decreased (Fig. 3 and Table III). 168 amino
acid residues are involved in this conformational change. This tertiary
structure change does not affect the conformation of the cytosolic
domains as illustrated by limited trypsin digestion experiments (Fig.
7). This idea is also supported by the fact that ATP hydrolysis
is not significantly modified in the presence of drugs (Fig. 11).
Consequently, the conformational change detected using H/D exchange may
reflect a restructuring of the protein in its membrane-embedded domain
where the drug(s)-binding site(s) are located. The fact that drug
binding does not affect the orientation of the transmembrane helices
may suggest a rotation or translation movement of the transmembrane
helices that would explain H/D exchange modifications. Interestingly,
cysteine cross-linking experiments on P-glycoprotein, another related
ABC transporter, have shown a reorganization of the membrane-embedded
helices after drug binding (59). However, in the case of Pgp, the
conformational change mediated by DOX and FCE binding to the membrane
helices is transmitted to the cytosolic domain and stimulates the
ATPase activity (60). These data suggest that, in the case of MRP1,
drug binding is not sufficient to allow the cross-talk between the
membrane and the cytosolic domains.
A drastic modification of the accessibility and/or the stability of the
conformational state of MRP1 is observed in the presence of GSH; the
proportion of unexchanged hydrogens increased from 39 to 60% (Fig. 3
and Table III). This conformational change affects, at least partly,
the conformation of the cytosolic domain as indicated by the protection
of the trypsin cleavage sites (Fig. 5) and could be required to
stimulate the ATPase activity (Fig. 11). These data strongly suggest
that GSH allows the coupling between drug transport and ATP hydrolysis.
This coupling cannot be achieved in the drug-bound state. It has been
reported previously that GSH binding greatly reduces
Km and increases Vmax for
drugs, suggesting a restructuring of the transmembrane helices that
might expose residues to the drug-binding site and increase the
affinity of the protein for the drug (9, 14). Our data suggest that
this change in affinity is related to rotation movements of
transmembrane helices along their own axis because the global tilt of
the transmembrane helices is not modified (Fig.
12, State III). These
rotation movements would reorient the amino acids involved in drug
binding in such a way that drug binding is enhanced.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-benzoylarginine-L-p-nitroanilide
(BAPNA) were purchased from Sigma. FCE
(3'-(3-methoxymorpholino)doxorubicin) was supplied by Farmitalia.
D2O was from Merck.
1 and encoded every 1 cm
1. The spectrophotometer was continuously purged with
air dried on a FTIR purge gas generator 75-62 Balston (Maidstone, UK)
at a flow rate of 5.8 liters/min. The internal reflection element (ATR)
was a germanium plate (50 × 20 × 2 mm) with an aperture angle of 45°, yielding 25 internal reflections (36).
1), which is
sensitive to the secondary structure (37). The analysis was performed
on the amide I region of deuterated samples because the H/D exchange
allows differentiation of the
-helical secondary structure from the
random secondary structure whose absorption band shifts from about 1655 cm
1 to about 1642 cm
1 (38). A combination
of Fourier self-deconvolution and a least squares iterative curve
fitting was applied to narrow and to quantify the area of the different
components of amide I band between 1700 and 1600 cm
1
(39). To avoid the introduction of artifacts because of the self-deconvolution procedure, the fitting was performed on the non-deconvoluted spectrum. The proportion of a particular structure was
computed as the sum of the area of all the fitted Lorentzian bands
having their maximum in the frequency region where that structure
occurs divided by the total area of the amide I. The frequency limits
for each structure were assigned according to theoretical (40) or
experimental (41) data: 1662 to 1645 cm
1,
-helix; 1689 to 1682 and 1637 to 1613 cm
1,
-sheet; 1644.5 to 1637 cm
1, random; 1682 to 1662.5 cm
1,
-turns.
1 equals zero on the dichroism spectrum,
to take into account the difference in the relative power of the
evanescent field for each polarization and also the differences in film
thickness as described previously (44).
) and
perpendicular polarization (A
) as shown in
Equation 1,
In ATR, the dichroic ratio for an isotropic sample
Riso is different from unity and is computed on the basis
of the area of the lipid ester band (1762-1700 cm
(Eq. 1)
1). As
described previously, the presence in the sample of a fraction x of oriented helices and a fraction 1
x
of randomly oriented dipoles results in an experimental dichroic ratio
RATR whose value is between the value of oriented helices
dichroic ratio R
and of randomly oriented dipoles Riso.
R
is calculated from x, Riso, and
RATR (36) as shown in Equation 2,
In an
(Eq. 2)
-helical structure, the amide I dipole is oriented
27° (44) with respect to the helix axis. The mean orientation of the
helix axis with respect to the membrane was estimated as described
previously (for review, see Ref. 45).
1. The signal from the
atmospheric water was subtracted as described by Goormaghtigh and
Russschaert (46). As described previously (40), deuteration of protein
side chains induces modifications in the amide I (1700 to 1600 cm
1) and amide II (1600 to 1500 cm
1)
regions. Several parameters modulate their contribution including the
ionization state of the carboxylic amino acids and the fraction of
deuterated and undeuterated amino acid side chains for every spectrum
of the kinetics. We used homemade software, which can compute the
contribution of the amino acid side chains as a function of the extent
of deuteration (47). The area of the amide II, characteristic of the
(N-H) vibration, was obtained by integration between 1596 and 1502 cm
1. For each spectrum, the area of the amide II was
divided by the corresponding lipidic
(C=O) area to take into account
small but significant variations in the total intensity due to the
presence of D2O which induced swelling of the sample layer
at the beginning of the kinetics (39). This ratio expressed in
percentage was plotted versus deuteration time. The value
corresponding to 0% of deuteration is defined by the amide II/lipid
ratio obtained before deuteration. The 100% value corresponds to a
zero absorption in the amide II region, observed for full deuteration
of the protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 region, is shown in Fig.
1. The lipid ester
(C=O) band is
located in the 1800 to 1700 cm
1 region. The amide I band
due to the
(C=O) vibration of the peptide bonds is located between
1700 and 1600 cm
1 and is highly sensitive to the
secondary structure. The 1600 to 1500 and 1500 to 1400 cm
1 bands represent unexchanged and exchanged amide II,
respectively, corresponding to the
(N-H) and
(N-D) amide
bonds.
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Fig. 1.
Infrared spectra in the 1800 to 1400 cm 1 region of MRP1 actively reconstituted into
lipids. Thin films were obtained by slowly evaporating a sample
containing 20 µg of MRP1 on an attenuated total reflection element.
Spectra were recorded as a function of the time (in minutes) of
exposure to D2O-saturated N2. Negative times
refer to spectra recorded before starting the deuteration procedure.
Inset shows the decrease in intensity of the amide II band
during the H/D exchange kinetics of MRP1.
MRP1 secondary structure composition determined in the absence and in
the presence of substrates
as(CH2) at 2920 cm
1 and
s(CH2) at 2851 cm
1 display a
negative deviation in the dichroic spectrum (Fig. 2C). This
means that the dipole is oriented perpendicular to the normal of the
germanium crystal plane. As the dipole orients perpendicular to the
hydrocarbon chains of lipids, the acyl chains are oriented normal to
the germanium plate.
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Fig. 2.
Polarized infrared and dichroism spectra of
MRP1. ATR-FTIR spectra of 20 µg of MRP1 recorded with incident
light polarized parallel (A) or perpendicular (B)
to the incidence plane. The dichroism spectrum (C) was
obtained by subtraction of the perpendicular polarized spectrum from
the parallel polarized one as described under "Experimental
Procedures."
1 indicating that
the oriented structure is essentially helical in nature because this
maximum is characteristic of
-helix structure and that the helices
are oriented mainly perpendicular to the germanium crystal plane,
i.e. perpendicular to the membrane plane. More quantitative
information upon the helix mean tilt requires the evaluation of the
dichroic ratio (R
) associated with the helical
structure. According to Equation 2, R
depends on
Riso, RATR, and x. x
represents the fraction of oriented helices. If we consider that only
the transmembrane helices are oriented with respect to the membrane and
taking into account that 22% of the amino acid residues of MRP1
corresponds to these transmembrane helices (17 transmembrane helices),
x = 22%. RATR = A
/A
, which is
the amide I dichroic ratio, and Riso, which is the dichroic
ratio of the randomly oriented dipoles (36, 43), are calculated as
described under "Experimental Procedures."
Mean orientation of helices obtained for MRP1 in the absence or in the
presence of different ligands
1) decrease as a
function of time of exposure to D2O-saturated N2 flow (Fig. 1). The decreasing area of amide II computed
between 100 and 0% (see "Experimental
Procedures") is reported in Figs. 3
and 4. In the absence of ligand, and
after 2 h of deuteration, 39% of the amide hydrogens remain
unexchanged. These amide hydrogens are poorly accessible to the aqueous
medium or involved in stable secondary structures. GSH binding strongly
affects the protein accessibility and/or the stability of the
conformational state of the protein because the percentage of
unexchanged amide hydrogens increased from 39 to 60% (Fig. 3). This
means that GSH binding mediates a conformational change that affects
21% (321 amino acid residues) of the total number of MRP1 amino acid
residues. Binding of FCE or DOX decreases the number of unexchanged
hydrogens from 39 to ~28%, indicating that a conformational change
has occurred that increases the accessibility of the protein toward the
aqueous phase and/or decreases the stability of the conformational
state of the protein (Fig. 3). 168 amino acids residues are implicated in this conformational change. In the presence of GSH and drug, no
significant change of the exchange rate is observed compared with the
protein alone, no matter what kind of drug was utilized (Fig. 3).
Around 40% of the amino acid residues remain unexchanged after 2 h of deuteration. This suggests that after simultaneous binding of GSH
and drugs, MRP1 behaves as in the absence of ligand in terms of
accessibility toward the aqueous phase.
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Fig. 3.
Evolution of the proportion of unexchanged
amide bonds of MRP1 computed between 100 and 0% as a function of the
deuteration time. , no substrate added;
, GSH;
, DOX;
, FCE;
, GSH and DOX; and
, GSH and FCE. The molar protein to
ligand ratio is 1:14 for DOX and FCE and 1:3 for GSH. The
curves are the means of three experiments.
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Fig. 4.
Evolution of the proportion of unexchanged
amide bonds of MRP1 computed between 100 and 0% as a function of the
deuteration time. , GSH and drugs;
, GSH, AMP-PNP, and DOX;
, GSH, ATP, and DOX;
, GSH, AMP-PNP, and FCE; and
, GSH, ATP,
and FCE. The molar protein to ligand ratio is 1:14 for DOX and FCE, 1:3
for GSH, and 1:6 for ATP or AMP-PNP. The curves are the
means of three experiments.
where ai is the proportion of each
amide group with identical Ti values.
(Eq. 3)
Proportion (a1, a2, a3) of the three
exponential components characterized by the period of T1 (1 min), T2 (9 min), and T3 (666 min)
Effect of ligands on trypsin activity
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Fig. 5.
Electrophoretic pattern of MRP1 peptides
after trypsin digestion in the absence or presence of GSH. MRP1
was treated without (lanes 2-6) or with 3 mM
GSH (lanes 7-11) for 10 min at 37 °C, followed by
trypsin digestion at a 1:400 (w/w) trypsin/protein ratio at room
temperature for the times indicated. The reaction was stopped by
addition of trypsin inhibitor (TLCK), and the tryptic fragments were
separated on a Tris-Tricine gel and visualized by silver staining. Each
lane contains 1.5 µg of protein. Lane 1 shows the protein
in the absence of trypsin.
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Fig. 6.
Topological model of human multidrug
resistance protein MRP1. A, MRP1 is thought to encode
for 17 putative transmembrane domains (TM) organized in
three membrane-spanning domains MSD0, MSD1, and
MSD2. The nucleotide-binding domains are indicated as
NBD1 and NBD2. L0 represents the
cytoplasmic loop linking MSD0 to MSD1; L1 represents the
cytoplasmic loop linking NBD1 to MSD2, and CL4 is the
cytoplasmic loop linking TM 7 to TM 8. B, the schematic
diagram indicates the approximate assignment of the tryptic fragments
described in the text.
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Fig. 7.
Electrophoretic pattern of MRP1 peptides
after trypsin digestion in the absence or presence of
anthracyclines. MRP1 was treated without (lanes 2-6)
or with 20 µM FCE (lanes 7-11) or 20 µM DOX (lanes 12-16) for 10 min at 37 °C,
followed by trypsin digestion at a 1:400 (w/w) trypsin/protein ratio at
room temperature for the times indicated. The reaction was stopped by
addition of trypsin inhibitor (TLCK), and the tryptic fragments were
separated on a Tris-Tricine gel and visualized by silver staining. Each
lane contains 1.5 µg of protein. Lane 1 shows the protein
in the absence of trypsin.
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Fig. 8.
Electrophoretic pattern of MRP1 peptides
after trypsin digestion in the absence or presence of anthracyclines
and GSH. MRP1 was incubated without ligand (lanes 2-6)
or in the presence of 20 µM DOX + 3 mM GSH
(lanes 7-11) or 20 µM FCE + 3 mM
GSH (lanes 12-16) for 10 min at 37 °C, followed by
trypsin digestion at a 1:400 (w/w) trypsin/protein ratio at room
temperature for the times indicated. The reaction was stopped by
addition of trypsin inhibitor (TLCK), and the tryptic fragments were
separated on a Tris-Tricine gel and visualized by silver staining. Each
lane contains 1.5 µg of protein. Lane 1 shows the protein
in the absence of trypsin.
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Fig. 9.
Electrophoretic pattern of MRP1 peptides
after trypsin digestion in the absence or presence of anthracyclines,
GSH, and MgAMP-PNP. MRP1 was treated without (lanes
2-6) or with 20 µM FCE + 3 mM GSH + 3 mM MgAMP-PNP (lanes 7-11) or 20 µM DOX + 3 mM GSH + 3 mM
MgAMP-PNP (lanes 12-16) for 10 min at 37 °C, followed by
trypsin digestion at a 1:400 (w/w) trypsin/protein ratio at room
temperature for the times indicated. The reaction was stopped by
addition of trypsin inhibitor (TLCK), and the tryptic fragments were
separated on a Tris-Tricine gel and visualized by silver staining. Each
lane contains 1.5 µg of protein. Lane 1 shows the protein
in the absence of trypsin.
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Fig. 10.
Electrophoretic pattern of MRP1 peptides
after trypsin digestion in the absence or presence of
anthracyclines, GSH, and MgATP. MRP1 was treated without
(lanes 2-6) or with 20 µM FCE + 3 mM GSH + 3 mM MgATP (lanes 7-11) or
20 µM DOX + 3 mM GSH + 3 mM MgATP
(lanes 12-16) for 10 min at 37 °C, followed by trypsin
digestion at a 1:400 (w/w) trypsin/protein ratio at room temperature
for the times indicated. The reaction was stopped by addition of
trypsin inhibitor (TLCK), and the tryptic fragments were separated on a
Tris-Tricine gel and visualized by silver staining. Each lane contains
1.5 µg of protein. Lane 1 shows the protein in the absence
of trypsin.
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Fig. 11.
Effect of anthracyclines and GSH on the MRP1
ATPase activity. ATP hydrolysis is presented as % of the control
ATPase activity assayed without ligand (8 nmol min 1
mg
1 of protein). A concentration of 20 µM
of each anthracycline and 3 mM GSH was used. The values
represented are the means of three experiments ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 12.
Schematic representation of different
structural states involved in GSH- and ATP-dependent drug
transport mediated by MRP1. The transmembrane domains are
represented by cylinders. The ellipse, hexagon,
and square represent the different conformations of the
cytosolic domain (NBDs are not represented). MRP1 has binding sites for
drugs (circle with hatched lines) and for GSH
(opened circle). These two sites may exist in a high
(circle with hatched lines and opened circle) or
low (square with hatched lines and opened square)
affinity state for their respective ligands (shaded for drug
and open for GSH). Drug and GSH are represented as
closed diamond and closed circle, respectively.
In and out refer to the inside and outside of the
cell. Arrows are representation of putative translation and
rotation movements of transmembrane helices. State I, the
high affinity binding sites of drug and GSH are exposed to the inner
leaflet of the bilayer for the drug and toward the cytosol for GSH.
State II, drug binding, in the absence of GSH, induces a
conformational change in the transmembrane domains which involves
rotational and/or translational movements. This conformational change
is not transmitted to the cytosolic domains. State III,
subsequent binding of GSH induces a structural reorganization affecting
both the cytosolic domains and the transmembrane domains. The
conformational change occurring in the cytosolic domain stimulates ATP
hydrolysis. The reorganization of the membrane-embedded domains of MRP1
involves rotational movements that reorients residues located in the
transmembrane helices and enhances drug binding. State IV,
ATP binding increases the accessibility of the cytosolic domain to the
aqueous phase. State V, ATP hydrolysis induces the rotation
of the transmembrane helices that decreases the affinity for the
ligands and allows their release into the external medium. State
IV', when a non-transported drug is bound to the protein, ATP
binding reorganizes the transmembrane domains through translational
movements. ATP hydrolysis (State V') modifies the tilt of
the transmembrane helices. Such a conformation limits the accessibility
toward the aqueous phase and restricts the translocation of the drug.
Our structural data provide no information about the mechanism by which
the protein comes back to its initial state.
Incubation of MRP1 with both GSH and drugs generates a conformation that is distinct from the conformation stabilized by each ligand. The level of H/D exchange is equivalent to the one obtained for the protein in the absence of ligand (Fig. 3 and Table III). Does this mean that GSH binding somehow compensates the effect of drug binding and brings the protein back into its initial conformation? This seems unlikely because limited proteolysis of MRP1 shows that simultaneous drug and GSH binding leads to a proteolytic pattern different from the one obtained with the protein alone (Fig. 8). In conclusion, in the presence of GSH and drug the protein adopts a conformation that does not correspond to that of the protein alone but both conformations are characterized by the same stability and/or global accessibility toward the solvent.
Conformational changes induced by binding and hydrolysis of ATP are analyzed in the presence of MgAMP-PNP and MgATP. The use of MgAMP-PNP, a non-hydrolyzable analog of MgATP, allows discrimination between the influence of nucleotide binding and nucleotide hydrolysis on MRP1 structure. The major structural reorganization of MRP1, during drug translocation, occurs after ATP binding, rather than ATP hydrolysis. ATP binding reorganizes MRP1 structure in a conformation that has a decreased stability and/or an increased accessibility to the aqueous phase (Fig. 4 and Table III). This conformational change affects at least partly the cytosolic domain as demonstrated by an increased sensitivity to trypsinolysis (Fig. 9). No significant change in the stability of this conformational state and/or the global accessibility of the protein (Fig. 4 and Table III) nor of the accessibility of the trypsin cleavage site (Fig. 10) is observed after ATP hydrolysis. It is possible that hydrolysis of ATP induces the rotation of transmembrane helices in the plane of the membrane. This movement does not require a change in the mean tilt of the transmembrane domains. The rotation of the transmembrane helices upon ATP hydrolysis has been demonstrated in the case of Pgp, by cysteine cross-linking studies (61). This rotation exposes different residues to the drug binding pocket, reducing the drug binding affinity. Furthermore, it has been shown previously that ATP hydrolysis, rather than ATP binding, reduces the affinity of MRP1 for its substrate (62).
It is also interesting to note that two-dimensional crystals of Pgp have revealed that ATP binding, not hydrolysis, drives the major conformational change associated with substrate translocation (63).
Binding of FCE, a non-transported drug, and GSH does not impair ATP binding and hydrolysis but affects the nature of the conformational change occurring during these steps of the transport process. ATP binding mediates a conformational change that decreases the accessibility of the protein to the aqueous phase and/or increases the stability of the conformational state (Fig. 4 and Table III). Subsequent hydrolysis of ATP does not modify the stability of this conformational state and/or the global accessibility of the protein. Despite this global decrease of accessibility, trypsin digestion reveals an increase of the accessibility of the cytosolic domain to the aqueous medium (Figs. 9 and 10). Consequently, the decrease in the global accessibility observed in H/D exchange upon binding and hydrolysis of ATP must affect the membrane domains. Interestingly, analysis of the dichroic spectra has revealed that a significant change in the mean orientation of the transmembrane helices occurs only upon ATP hydrolysis. This supports the idea that after ATP binding, the reorganization of the transmembrane helices involves translational movements, whereas ATP hydrolysis modifies the tilt of the transmembrane helices. This reorganization inhibits the passage of the drug from its high affinity binding site to its low affinity binding site, and the drug remains bound to the protein.
Translocation Model Proposed for MRP1-- A model describing the MRP1-mediated mechanism of drug transport is proposed in Fig. 12. This model is based on the biochemical evidence that drugs binding to the protein occur from the inner leaflet of the bilayer (26-32). In contrast, GSH-binding site is accessible from the cytosol (64). ATP hydrolysis reduces the affinity of MRP1 for its substrate (61), which allows its release into the extracellular medium (13, 18). In its initial state (Fig. 12, State I), the high affinity drug-binding site is exposed to the membrane, and the high affinity GSH-binding site is oriented toward the cytosolic domain. The protein-drug interaction causes a structural modification in the membrane-spanning domains that may involve rotation or translation movements (Fig. 12, State II). Subsequent binding of GSH (Fig. 12, State III) modifies the structure of the membrane-embedded domain and the ATP-binding sites located in the cytosolic domain. The restructuring of the transmembrane helices may be due to rotation movement in the membrane but not to a reorientation of these domains. The cytosolic domain adopts a conformation that stimulates ATP binding and/or hydrolysis. When a transported drug (DOX) is bound to the protein, ATP binding (Fig. 12, State IV) causes a conformational change which opens a pathway through which translocation occurs, a prerequisite for the release of the substrates into the extracellular medium. ATP hydrolysis (Fig. 12, State V) would change the affinity of MRP1 for substrate through helix rotation in the drug-binding domain. Consequently, they are released into the external medium. Occupation of the binding site by a non-transported drug (FCE) (Fig. 12, State IV' and State V') may prevent it from gaining access to the opposite side of the membrane and thereby prevent transport.
In conclusion, we have characterized several structural intermediates
involved in MRP1-mediated drug transport. It is worth mentioning that
structural changes, first identified by using the IR approach described
here, have been shown to be consistent, respectively, with a
three-dimensional map and two-dimensional crystals for
H+-ATPase (65, 66) and Pgp (63, 67).
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of Alice and David van Buuren Fellowship and Fonds pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture.
To whom correspondence should be addressed: Laboratoire de
Structure et Fonction des Membranes Biologiques, Free University of
Brussels, CP 206/2, Bd. du Triomphe, B-1050 Brussels, Belgium. Fax:
30-2-6505382; E-mail: jmruyss@ulb.ac.be.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M207963200
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ABBREVIATIONS |
---|
The abbreviations used are:
MRP1, multidrug
resistance protein 1;
Pgp, P-glycoprotein;
NBD, nucleotide binding
domain;
TM, transmembrane segments;
FCE, 3'-(3-methoxymorpholino)doxorubicin;
DOX, doxorubicin;
AMP-PNP, 5'-adenylyl--
-imidodiphosphate;
ATR-FTIR, attenuated total
reflection Fourier transform infrared;
H/D, hydrogen/deuterium;
ABC, ATP-binding cassette;
BAPNA, N-
-benzoylarginine-L-p-;
TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
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