From the Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology,
Manchester M60 1QD, United Kingdom, ¶ Cancer Research
Laboratories, Queen's University, Kingston, Ontario K7L 3N6,
Canada, and the
Electron Microscopy Center and Department of
Biology, Texas A&M University, College Station, Texas
77843-2257
Received for publication, January 9, 2001
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ABSTRACT |
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Multidrug resistance protein 1 (MRP1/ABCC1) is an
ATP-binding cassette (ABC) polytopic membrane transporter of
considerable clinical importance that confers multidrug resistance on
tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other
ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a
nucleotide binding domain (NBD). However, unlike P-glycoprotein and
most other ABC superfamily members, MRP1 contains a third MSD with five
predicted transmembrane segments with an extracytosolic NH2 terminus. Moreover, the two nucleotide-binding
domains of MRP1 are considerably more divergent than those of
P-glycoprotein. In the present study, the first structural details of
MRP1 purified from drug-resistant lung cancer cells have been obtained
by electron microscopy of negatively stained single particles and
two-dimensional crystals formed after reconstitution of purified
protein with lipids. The crystals display p2 symmetry with
a single dimer of MRP1 in the unit cell. The overall dimensions of the
MRP1 monomer are ~80 × 100 Å. The MRP1 monomer shows some
pseudo-2-fold symmetry in projection, and in some orientations of the
detergent-solubilized particles, displays a stain filled depression
(putative pore) appearing toward the center of the molecule, presumably
to enable transport of substrates. These data represent the first
structural information of this transporter to ~22-Å resolution and
provide direct structural evidence for a dimeric association of the
transporter in a reconstituted lipid bilayer.
The 190-kDa multidrug resistance protein
MRP11 (ABCC1) is a polytopic
membrane transport protein that belongs to the ATP-binding cassette
(ABC) superfamily and has been detected in many different drug-resistant cell lines and tumor tissues since it was first cloned
in 1992 (1-6). When overexpressed in tumor cells, MRP1 confers
multidrug resistance by reducing intracellular drug concentrations in
an ATP-dependent manner (6-8). In this respect, MRP1 is
similar to another ABC transporter, the well characterized 170-kDa
P-glycoprotein (P-gp) (ABCB1) (9, 10). ABC proteins play important
physiological and protective functions in bacteria, yeast, plants, and
mammals and are capable of transporting a wide variety of molecules
across biological membranes. Known substrates for ABC transporters
include ions, phospholipids, steroids, polysaccharides, amino acids,
peptides, and in the case of several MRP-related proteins, anionic
conjugated endo- and xenobiotics (6, 10-12). In addition to MRP1 and
P-gp, other examples of clinically important human ABC proteins include the cystic fibrosis transmembrane conductance regulator CFTR (13), and
the sulfonylurea receptor (SUR), which is part of an
ATP-sensitive potassium channel involved in insulin secretion (14).
The amino acid sequence of MRP1 predicts that it contains a core
structure that is common to many ABC proteins, namely two hydrophobic
membrane-spanning domains (MSDs) each followed by a nucleotide binding
domain (NBD) (1). Most current evidence suggests that these two MSDs
each consist of six transmembrane Transport studies using both MRP1-enriched plasma membrane vesicles and
purified reconstituted MRP1 have established that MRP1 can actively
transport a structurally diverse array of conjugated organic anions,
including the cysteinyl leukotriene LTC4 and oxidized glutathione (6, 12, 22-24). In contrast, studies using MRP1-enriched plasma membrane vesicles under similar conditions have failed to
demonstrate that MRP1 directly transports unmodified chemotherapeutic agents (12, 26, 27). However, recent studies suggest that certain
unconjugated drugs such as vincristine and daunorubicin are actively
co-transported by MRP1 with reduced glutathione (25-27). These
transport properties of MRP1 are quite different from those described
for P-gp, and the structural features responsible for these differences
between the two drug resistance proteins are not yet known. Indeed,
little is known about the structure of MRP1.
We recently reported immunoaffinity purification of native MRP1 from
the highly drug-resistant lung cancer cell line, H69AR, from which MRP1
was originally cloned (1, 24, 28). The purified reconstituted native
protein exhibits low level basal ATPase activity that is stimulated by
its organic anion substrates (28). More recently, we have demonstrated
ATP-dependent transport of LTC4 and other
substrates in an artificial lipid bilayer system containing purified
MRP1 (24). These data support the concept that MRP1 functions as an
energy-dependent efflux pump, which couples translocation
of its substrates with ATP hydrolysis. To understand the molecular
mechanisms by which MRP1 recognizes and transports its substrates, and
how this is coupled to ATP hydrolysis, it is desirable to obtain high
resolution structural information on the protein. We have previously
reported the low resolution structure of P-gp (29, 30), as determined
by electron microscopy of single particles (31) and small epitaxial
two-dimensional crystals and more recently, from larger two-dimensional
crystals2 by electron
crystallography (32). In the present paper, we have obtained structural
data for MRP1 up to ~22-Å resolution using similar approaches and
describe the first two-dimensional crystals of this transporter in the
presence of phospholipids. Analysis of the two-dimensional crystals in
negative stain suggests that MRP1 acts as a dimer. This structural
information provides a basis for further mechanistic studies of MRP1
and related ABC proteins.
Materials--
Copper electron microscope grids and uranyl
acetate were purchased from Agar Scientific (Essex, UK). Dimyristoyl
L- Purification of MRP1--
Plasma membranes were prepared as
described previously from the doxorubicin-selected multidrug-resistant
small cell lung cancer cell line H69AR (25). Plasma membranes
containing MRP1 were solubilized with CHAPS and subjected to
immunoaffinity chromatography as described in a previous study (24).
Purity was estimated by densitometric scanning of polyacrylamide gels
stained with alkaline silver and by immunoblotting (24) and only
preparations of Single-particle Analysis of Negatively Stained
MRP1--
Purified MRP1 (12 µg ml Two-dimensional Crystals of MRP1 and Image
Processing--
Purified MRP1 (~100 µg ml Purification of MRP1--
MRP1 purity was judged to be greater
than 90% based on the following criteria: (i) detection of a single
band in an alkaline silver-stained gel of the protein resolved by
polyacrylamide gel electrophoresis (Fig.
1), (ii) its homogeneity, as assessed by electron microscopy (Fig. 2), and (iii)
its ability to crystallize (Fig. 5). ATPase and transport assays of the
reconstituted purified protein confirmed that it was active and its
activity could be inhibited by conformation-dependent
MRP1-specific monoclonal antibodies (24, 28, 39). To obtain initial
structural data for detergent-solubilized MRP1, samples were analyzed
by electron microscopy of single particles.
Single-particle Analysis of MRP1--
Fig. 2 shows a transmission
electron micrograph of a negatively stained preparation of purified
MRP1 in DDM (panel a) and a montage of representative MRP1
molecules (panel b) (the protein appears as light
shades and stain as dark shades). The visualized particles were orientated differently with respect to the incident electron beam, but the high degree of homogeneity of the protein was
shown by the consistent size of the protein molecules, with most
molecules having a maximal diameter of ~100 Å. Rotational and
translational alignment followed by multivariate statistical analysis
of the images of selected particles enabled differences in the images
to be analyzed. This allowed the particles to be classified according
to the orientation of the particle (e.g. top and
side views), which were separated by correspondence analysis followed by hierarchical ascendant classification (29, 31). Images that
were similar were merged together to form similar projections or
classes (35, 36) and after further rounds of rotational and
translational alignment were finally averaged. The class averages thus
showed the most commonly occurring projections with a concomitant
improvement in the signal-to-noise ratio. This algorithm was
implemented using SPIDER (31, 34) using a reference-free-based alignment method (35, 36), which offers the advantage of the final
image not being dominated by a chosen reference. The assignment into
various classes of projection at a cutoff level of 0.34 is represented
by the dendrogram shown in Fig.
3. Various projections are shown by
classes I-V. Each of the two major classes (I and IV) had a good
within-group resolution (22 Å) as judged by Fourier Ring Correlation
(31, 34). There appears to be mainly face-on views of MRP1 with side-on
views (where the molecule would be likely to display a rectangular
appearance perpendicular to the incident electron beam) appearing less
frequently probably due to preferred orientations of MRP1 on the grid
surface. The three projection maps corresponding to the three most
populous classes (I, II, and IV) are shown in Fig.
4a, b, and
c, respectively.
Fig. 4 (a and c) shows that the predominant
projection of MRP1 was toroidal in shape similar to that observed
earlier for P-gp (29) with a ring of protein comprised of five or six
densities surrounding a central stain filled region of ~35-Å
diameter. The handedness of these class averages was thought to be an
"up" view (see below). Some protein densities appear to be roughly
related by pseudo-2-fold symmetry, although the overall shape of this average is approximately pentagonal. A rotational autocorrelation match
and rotational filtering, implemented by RFILTIMPER in MRC (see below),
suggested there was some 2- and 5-fold symmetry in MRP1 (data not
shown). The averaged projection structure shows a relatively open
arrangement of protein densities surrounding the outer perimeter. Fig.
4b shows a view, which appears distinctive from Fig. 4
(a and c) and is calculated from relatively fewer particles, and hence the resolution was ~35 Å. The
asterisks show two densities related by 2-fold
pseudosymmetry. The third NH2-terminal MSD of MRP1 might be
expected to reduce any pseudo-2-fold symmetry (40). An indication from
these projection maps for such behavior is in Fig. 4b where
the density labeled 3 sits somewhat apart from the densities
1, 2, 4, and 5. Density
3 in Fig. 4a also remains apart from its
neighboring densities, whereas density 4 protrudes more from
the perimeter of the protein ring.
Two-dimensional Crystals of MRP1--
Because of the high degree
of homogeneity of the purified MRP1, attempts were made to grow
two-dimensional crystals of the protein by Bio-beads-assisted detergent
removal at a suitable ratio of protein to lipid. This allowed us to
establish whether a higher resolution structure of MRP1 would be
attainable by electron cryomicroscopy. Fig.
5 shows a small two-dimensional crystal
of MRP1 with lattice dimensions 133 × 256 Å. Low dose images of
vesicles from several crystallization trials showed two-dimensional
arrays displaying order to ~25 Å as judged by optical diffraction.
After correction for lattice distortions using image processing,
structure factors were found to ~20-Å resolution. A typical plot of
a computed Fourier transform for one of the crystals is depicted in
Fig. 6 with high signal-to-noise (IQ 3)
(41, 42) reflections detected up to a resolution of ~20 Å. Initial
analysis on the basis of phase comparisons suggested p2
symmetry could be present (37). Projection maps of MRP1 crystals, with
data truncated at ~25-Å resolution with no symmetry (p1)
(Fig. 7a) and with
p2 symmetry enforced (Fig. 7b) are shown after
merging data from three independent lattices. In Fig. 7b the
putative monomer is outlined. The unit cell appears to contain two MRP1
molecules, related by a crystallographic 2-fold axis perpendicular to
the membrane. The molecular boundaries of a monomer (outlined) were
apparent. The projection map suggests that the intermonomer contacts
are more extensive on one side of MRP1. The projection map appears
distinct from those calculated by single-particle analysis, and it is
possible to explain these differences in accordance with stain
distribution at the intracellular as opposed to the extracellular
surface for the single-particle map (see "Discussion"). The quality
of the merged projection map can be judged from the statistics shown in
Table I. The overall p2 phase
residual to 20-Å resolution was 21° (where 45° is random or non
p2 phases) providing confidence in the structure obtained and the plane group assignment.
Since the cloning of MRP1 from the human small cell lung cancer
cell line H69AR, expression of this ABC transporter has been widely
detected in both resistant tumor cell lines and clinical samples (1, 6,
12) similar to that observed for P-gp (10). Here, we report the first
projection structure of MRP1 to ~22-Å resolution determined by
single-particle analysis and electron crystallography. The MRP1 used in
this study was purified from H69AR lung cancer cells and retains its
ATPase and transport activities as well as its reactivity with
antibodies that recognize conformation-dependent epitopes,
demonstrating that it remains folded in an active configuration (24,
28, 39).
In the present study MRP1 appears to be monomeric after detergent
solubilization and isolation but dimeric upon reconstitution into
crystalline protein/lipid arrays (Fig. 7), which may have important
functional implications. Whether MRP1 is dimeric in native membranes is
not known at present, because the dimer observed in these projection
maps may simply reflect favorable crystallographic interactions.
However, earlier radiation inactivation studies have suggested MRP1 can
function as a dimer in the membrane (43). The cystic fibrosis
transmembrane regulator (CFTR), which belongs to the same subfamily C
of the ABC transporters as MRP1, also appears to possess a dimeric
character when bound to a recombinant CAP70 protein (44). A dimeric
association for CFTR has been further implied from experiments using a
fusion protein consisting of two tandemly linked CFTR coding sequences
(45) and by electron microscopy studies (46). However, other
biochemical studies using monoclonal antibodies have proposed a
monomeric form for CFTR (47). Structural studies for P-gp, which is
structurally more dissimilar to MRP1 than CFTR, have shown that it
exists as a monomer as both single particles and in two-dimensional
crystals, although the crystals contained no lipid
(29).2
The single-particle images could readily pack into the crystal lattice
shown in Fig. 7, and the size of the MRP1 monomer from the
two-dimensional crystals closely matched the longest dimension (100 Å)
for the single particles along b, whereas the dimensions along a were slightly smaller (70 Å). Negative stain,
however, is unlikely to penetrate the sealed liposomes and,
consequently, projection maps will tend to be dominated by the protein
domains exposed on the outer surface of the liposomes. Thus, if the
protein domains are only partially exposed along a, then
this may account for the difference along this direction. For the
detergent-solubilized MRP1, a toroidal ring of protein surrounds a
large (35 Å in diameter) stain-filled region, which is not detected
for the crystals. This could be explained, if the view for the single
particles represents predominantly the extracellular surface of the
molecule and the two-dimensional crystals shows the intracellular
surface exposed outside the liposomes, which is dominating the
projection. Reconstitution experiments by Manciu et al. (48)
using similar conditions to those described here showed that MRP1
inserted into liposomes in an inside-out configuration, with its
catalytic sites or NBDs outside the liposomes. These findings suggest
that the projection maps derived from the two-dimensional crystals
could be representative of the stain distribution at the intracellular
surface of MRP1 (Fig. 7). The monomer appears to contain 2 pseudosymmetrical domains that could be consistent with this
interpretation. Similar features are observed for the intracellular
surface of P-gp crystals, which showed two pseudosymmetrical densities
separated by ~90 Å. These were assigned as the NBDs because the
x-ray crystal structure of the NBDs of histidine permease (49) could be
comfortably docked with them.2 In contrast, the
extracellular surface of P-gp is characterized by a toroidal ring of
protein densities with a large central depression, similar to that
observed for MRP1. Because the extracellular surface of MRP1 could
associate preferentially with the specimen support film, possibly
because of its surface charges, this region could be the most strongly
stained, and thus it is feasible that features on this side of the
molecule could dominate a projection from single particles. Our
preliminary interpretation is that MRP1 is structurally similar to P-gp
(29),2 with a large extracellular pore surrounded by a ring
of protein, with the pore sealed on the intracellular side by its two
NBDs. This working model will be tested in the future by electron
cryomicroscopy of unstained specimens and by three-dimensional reconstructions.
Several MRP-related ABC proteins that have been characterized are
organic anion transporters, and thus their relatively hydrophilic substrates do not readily penetrate the cell membrane. The large putative pore in MRP1 might facilitate transport of these charged substrates. The putative pore opens at the extracellular face of the
membrane, but it vastly exceeds the diameter required for the passage
of known MRP1 substrates and opening of such a pore across the membrane
would obviously not be compatible with cell viability. However, our
studies from P-gp and now from MRP1 two-dimensional crystals suggest
that the pore of this protein is closed at the cytoplasmic face of the
membrane by either the intracellular hydrophilic loops and/or the NBDs
(29).2 The NBDs of MRP1 could serve a similar function,
although it should be borne in mind that the two NBDs of MRP1 are
considerably more divergent than those of P-gp and appear to be more
functionally distinct with respect to their ATP binding and hydrolysis
properties (50-52).
The various projection maps appear to suggest that MRP1 exhibits some
2-fold pseudosymmetry, which may be explained by the core structure
comprising two MSDs each consisting of six transmembrane segments and
two NBDs. This was supported by the rotational autocorrelation search
and rotational filtering for the single-particle-derived data and the
two-dimensional crystals, respectively, described earlier. The
NH2-terminal MSD1 comprised of five transmembrane segments
found in MRP1 might be expected to cause the protein to deviate from
2-fold pseudosymmetry, and this is supported by the observation of some
5-fold pseudosymmetry in the autocorrelation search for the
single-particle data. The protrusion on density 4 and the
additional density 3 (Fig. 4) also supports a deviation from
2-fold pseudosymmetry. Fig. 8 shows a
comparison of solubilized P-gp and MRP1 structures in projection,
truncated at approximately the same resolution (22 Å) where the major
differences are highlighted (see arrows). It is possible to
speculate that density 3 and/or 4 (Fig. 4) might
correspond to the MSD1 of MRP1 and this requires further investigation.
For example, previous studies have shown that MRP1, which has been
NH2-terminally truncated at amino acid position 204, and
thus lacking MSD1, retains its ability to transport LTC4
(21). It could be of future interest to determine the structure of this
truncated MRP1 to establish how it compares with that of the
full-length protein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (15-18) and that the two
NBDs are located on the cytoplasmic face of the plasma membrane (6,
19). In addition to this core, four-domain structure is a third,
NH2-proximal MSD (MSD1) of ~200 amino acids with an
extracytosolic NH2 terminus that is thus far found only in
MRP1, SUR, and several other members of ABC subfamily "C" (1, 6,
12, 17). Certain modifications of MSD1 and the intracellular loop
connecting it to MSD2 can inactivate the protein (20, 21), suggesting
that interaction of MSD1 with other domains may play an important role
in the regulation of MRP1 activity.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-phosphatidylcholine was from Sigma (Gillingham,
Dorset, UK) and n-dodecyl-
-D-maltoside (DDM)
was from Calbiochem (Nottingham, UK). Bio-Beads were obtained from
Bio-Rad (Hertfordshire, UK).
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)
was from ICN (Aurora, OH).
90% purity were used for subsequent electron
microscopy. In addition, all preparations of purified MRP1 used were
reconstituted into proteoliposomes and tested for ATPase activity and
transport activity as described previously (24, 28).
1) was applied to
freshly glow-discharged 400-mesh copper/carbon grids for 30 s and
negatively stained in 4% w/v uranyl acetate (1 min). Scattered
electrons from the stain dominate the images from negative staining,
and thus, the protein is seen in negative. However, negative stain can
readily provide information on the size, shape, and oligomeric form of MRP1. Electron micrographs were recorded at calibrated magnifications in a Philips CM 100 and a Tecnai 10-transmission electron microscope operating at 100 kV under low dose conditions. Images were recorded on
Agfa Scientia film and developed for 4 min in PQ developer. The quality
of the micrographs was checked on an optical diffractometer for
astigmatism and drift, and suitable films were digitized at 4.2 Å per
pixel at the specimen level using a Zeiss SCAI microdensitometer. Six
hundred well- separated particles were interactively selected using
Ximdisp, which comprises part of the MRC image-processing suite (33).
Images were processed using the SPIDER image-processing software (31,
34) with reference-free alignment (35). Images were initially
low-pass-filtered (filtering low resolution information) using a
Gaussian band-pass filter to ~27-Å resolution, and the shifts and
rotations were applied to the unfiltered images. The alignment
converged after six rounds producing only a small change in the
alignment parameters. The images were subjected to correspondence analysis and classification (36) with each class representing a
relatively homogeneous subgroup of images. Each class was aligned separately, and average images were calculated by summing the aligned
particles within one appropriate class (29). Symmetry was tested by a
rotational autocorrelation search.
1) was
reconstituted with dimyristoyl L-
-phosphatidylcholine at
a protein/lipid ratio of 0.5-1.0 (w/w) with lipids solubilized in
0.1% (w/v) DDM in 50 mM Tris-HCl (pH 7.5) buffer
containing 3 mM NaN3. The detergent was removed over 6 h using SM2-Bio-Beads (15 g 100 ml
1) at
22 °C. Electron microscope grids were prepared as above by negative
staining by allowing 5 µl of the reconstituted vesicles to sit for 1 min on a 400-mesh glow-discharged carbon-coated copper grid. Excess
solution was removed, and the specimen was negatively stained with 4%
(w/v) uranyl acetate (1 min) and blotted. Patches of two-dimensional
crystals (unit cell ~ 139 × 256 Å) were observed in some
vesicles after 6-h incubation with Bio-Beads. Controls with protein
omitted showed no crystals. Images were recorded and scanned as
described above. Images were screened using an optical diffractometer
for drift and astigmatism and to check for crystalline arrays of MRP1.
Processing of images by correcting for the distortions in the crystal
lattice (lattice unbending) and effects of the contrast transfer
function and astigmatism was performed using the MRC-LMB software (33).
Comparison of phase residuals (37) suggested p2 symmetry and
is discussed below. Projection maps of the merged data were calculated
using the standard crystallographic computer programs in the CCP4
package (38).
RESULTS
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Fig. 1.
Polyacrylamide gel electrophoresis of
solubilized immunoaffinity-purified native MRP1. One microgram of
purified native MRP1 was subjected to electrophoresis on a 7.5% w/v
polyacrylamide gel and detected by alkaline silver staining. Molecular
mass markers (kDa) are on the left.
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Fig. 2.
Electron microscopy of CHAPS-solubilized MRP1
in uranyl acetate. a, the majority of the particles
were ~100 Å in diameter. b, galleries of typical
low-pass-filtered molecules, to ~35-Å resolution, shown at higher
magnification, which were selected for single-particle analysis. A
stain-filled cavity is evident in some of the molecules. The
scale bar in a shows 500 Å and in b
shows 250 Å.
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Fig. 3.
Definition of particle classes after
single-particle analysis. A linkage tree was obtained from
hierarchical ascendant classification using complete linkage as a
merging criterion. The cutting level was 0.34, which generated a
suitable within-group resolution of the major classes (see
"Results"). The number of particles within each class is indicated
above the images.
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Fig. 4.
Projection maps of solubilized MRP1
calculated by single-particle analysis. Images of 600 particles
were classified into subgroups using SPIDER. The stain is delineated in
black and the protein in white. The three major
classes of MRP1 particles (a-c), which differ in their
orientation with respect to the electron beam, are shown. The
scale bar represents 39 Å.
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Fig. 5.
Electron micrograph of two-dimensional
crystals of dimyristoyl
L- -phosphatidylcholine vesicles
containing MRP1 (1:1, protein:lipid, w/w) stained with 2% w/v uranyl
acetate. The white arrows indicate the two-dimensional
crystal. The scale bar corresponds to 500 Å.
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Fig. 6.
A computer-generated Fourier transform of an
image of a single two-dimensional crystal. The data shown was
obtained after three cycles of lattice unbending, which compensates for
distortion in the crystal lattice by applying the lattice vectors of an
ideal lattice usually selected from the center of the crystal to the
remainder of the crystalline area. The numbers in the
boxes indicate the quality of the reflection with
large squares and low numbers indicating
reflections of high signal-to-noise ratio (IQ) (41, 42). The IQ value
represents the signal-to-noise above background with a lower IQ value
(e.g. 1-4) representing a good signal-to-noise
ratio of the reflection whereas a higher IQ value (e.g.
7-8) represents a poor one. The arrows indicate
the direction of the reciprocal lattice vectors (h and
k), and the circles represent zero values in the
contrast transfer function. The edge of the plot corresponds to a
resolution of ~20 Å.
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Fig. 7.
Projection maps of MRP1 in negative stain at
25-Å resolution calculated from images of two-dimensional crystals of
MRP1. In a no symmetry (p1) and in
b p2 symmetry is enforced. Data from a single
crystal (a) and data from amplitudes and phases merged from
three independent lattices (b) are shown. One unit cell
contains two molecules of MRP1. The solid lines indicate
density above the mean with contours extending up to 1.5 sigma in six
even steps; dotted lines indicate density below the mean
value. A putative monomer is shown by the dotted lines. In
a, 2-fold axes of symmetry are evident. In b, the
phases were constrained to 0° or 180°, and the projection is
centrosymmetric. The scale bar corresponds to 30 Å.
Crystallographic data for negatively stained images of MRP1
DISCUSSION
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Fig. 8.
Comparison of solubilized MRP1
(a) and P-gp (b) projection
structures predicted to correspond to the extracellular face of the
proteins (see "Discussion"). Both structures were filtered to
the same resolution and similarly contoured to facilitate a comparison
between the two. The projection in a corresponds to the
class shown in Fig. 4a. P-gp data are from previous studies
(29, 30). The scale bar corresponds to 22 Å.
In conclusion, this work represents the first structure of a protein
from the MRP subfamily C of the ABC transporter superfamily that
differs in its general architecture from that of P-gp. Dimeric characteristics of MRP1 appear after reconstitution into lipid bilayers, although its in vivo oligomeric form is still not
known. Our data are in agreement with most of the biochemical evidence available for MRP1. This structural data should contribute to a greater
understanding of the substrate binding and transport mechanism of MRP1
and related ABC transporters.
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ACKNOWLEDGEMENTS |
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We are grateful to the Royal Society (London) for a travel grant (to M.F.R.) and to Dr. A. Kitmitto and Giles Velarde (University of Manchester Institute of Science and Technology) for useful discussions.
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FOOTNOTES |
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* Supported in part by the Medical Research Council of Canada (Grant MT-10519).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 a fellowship from the Royal Society (London, UK). To whom correspondence should be addressed: Tel.: 44-161-200-4186; Fax: 44-161-236-0409; E-mail: mark.rosenberg@umist.ac.uk.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M100176200
2 M. F. Rosenberg, G. Velarde, R. C. Ford, C. Martin, G. Berridge, I. D. Kerr, R. Callaghan, A. Schmidlin, C. Wooding, K. J. Linton, and C. F. Higgins, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are:
MRP1, multidrug
resistance protein 1;
P-gp, P-glycoprotein;
ABC, ATP-binding cassette;
MSD, membrane-spanning domain;
NBD, nucleotide-binding domain;
LTC4, leukotriene C4;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CFTR, cystic fibrosis transmembrane regulator;
SUR, sulfonylurea receptor;
DDM, n-dodecyl--D-maltoside;
IQ, signal-to-noise ratio.
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