(Received for publication, October 25, 1996, and in revised form, February 4, 1997)
From the Department of Biochemistry and Applied
Molecular Biology, University of Manchester Institute of Science
and Technology, P. O. Box 88, Manchester M60 1QD, United Kingdom
and ¶ Nuffield Department of Clinical Biochemistry and Imperial
Cancer Research Fund Laboratories, Institute of Molecular Medicine,
University of Oxford, John Radcliffe Hospital,
Oxford OX3 9DU, United Kingdom
P-glycoprotein (P-gp) is a member of the ATP
binding cassette superfamily of active transporters and can confer
multidrug resistance on cells and tumors by pumping chemotherapeutic
drugs from the cytoplasm. P-gp was purified from
CHrB30 cells and retained the ability to bind
substrates and hydrolyze ATP. Labeling of P-gp with lectin-gold
particles suggested it is monomeric. An initial structure of purified
P-gp was determined to 2.5 nm resolution by electron microscopy and
single particle image analysis of both detergent-solubilized and
lipid-reconstituted protein. The structure was further refined by three
dimensional reconstructions from single particle images and by Fourier
projection maps of small two-dimensional crystalline arrays (unit cell
parameters: a, 14.2 nm; b, 18.5 nm; and ,
91.6°). When viewed from above the membrane plane the protein is
toroidal, with 6-fold symmetry and a diameter of about 10 nm. There is
a large central pore of about 5 nm in diameter, which is closed at the
inner (cytoplasmic) face of the membrane, forming an aqueous chamber
within the membrane. An opening from this chamber to the lipid phase is
present. The projection of the protein perpendicular to the membrane is
roughly rectangular with a maximum depth of 8 nm and two 3-nm lobes
exposed at the cytoplasmic face of the membrane, likely to correspond to the nucleotide binding domains. This study provides the first experimental insight into the three-dimensional architecture of any ATP
binding cassette transporter.
The multidrug resistance P-glycoprotein (P-gp)1 is an active transporter located in the plasma membranes of many cells and tissues (1). P-gp has a relatively broad specificity for hydrophobic compounds. Although its natural substrate(s) is not known, when overexpressed P-gp can confer multidrug resistance on cells and tumors by pumping chemotherapeutic drugs from the cytoplasm. P-gp is a member of the ATP binding cassette (ABC) superfamily of transporters and channels (2). More than 100 ABC transporters have been identified in bacteria, yeasts, plants, and mammals, including the cystic fibrosis and adrenoleukodystrophy gene products, the pfmdr gene product associated with drug resistance of the malarial parasite, and the TAP (ransporter associated with ntigen resentation) peptide transporter, which is essential for antigen presentation. ABC transporters share a common domain organization and considerable amino acid sequence identity, implying a common architecture and evolutionary origin.
P-gp is a 170-kDa polypeptide that is glycosylated at a single site at
the extracellular face of the membrane. From its primary sequence, P-gp
is predicted to consist of four domains. Two hydrophobic transmembrane
domains each consist of six membrane-spanning segments (putative
-helices) separated by hydrophilic loops (3, 4). These transmembrane
domains are believed to form the pathway through which solute crosses
the membrane and to play a major role in determining substrate
specificity. The other two domains, the nucleotide binding domains
(NBDs), are located at the cytoplasmic face of the membrane and couple
ATP hydrolysis to the transport process (5, 6).
Although a considerable body of biochemical and genetic data has accrued for many ABC transporters, little is known about the three-dimensional organization of these proteins. Indeed, the absence of any structural data is the principal factor that limits our understanding of the molecular mechanisms of active transport. In this study we present an initial structure for P-gp, determined to 2.5 nm resolution. These data provide the first structural insights for any ABC transporter.
Chinese hamster
ovary CHrB30 cells were grown and maintained in a medium
containing 30 µg/ml colchicine as described previously (7). Plasma
membranes from these cells were prepared according to the method of
Lever (8) with minor modifications (7). Membranes were stored at a
protein concentration of 5-7 µg/µl at 80 °C for up to 6 months without loss of P-gp activity.
Membranes from
CHrB30 cells were solubilized in the mild, nonionic
detergent dodecyl maltoside and purified by anion exchange and
hydroxyapatite chromatography as described in detail
elsewhere.2 Fractions containing P-gp from
the final hydroxyapatite chromatographic step were concentrated to
approximately 0.2 µg/µl and stored at 20 °C.
Large unilamellar liposomes of asolectin lipids were generated at a
lipid concentration of 15 mg/ml in dialysis buffer (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl) as described previously
(10). The liposomes were solubilized with 40 mM
octyl--glucoside and added to purified P-gp at a lipid:protein ratio
of 10:1 (w/w) for 30 min at 4 °C. Detergent was subsequently
removed, and P-gp was reconstituted into asolectin lipids by Sephadex
G-50 gel filtration chromatography (1 × 60 cm) using 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, elution
buffer. The void volume fractions were collected, and proteoliposomes
were concentrated by centrifugation at 150,000 × g for
4 h at 4 °C. The efficiency of reconstitution was assessed by
the co-migration of P-gp with tracer
[3H]phosphatidylcholine during sucrose density
centrifugation (11).2
ATP hydrolysis by purified, reconstituted P-gp was determined essentially as described (12, 13). Each assay was carried out in a total volume of 150 µl of assay buffer (150 mM NH4Cl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.02% NaN3) containing 0.5 µg of protein and 2 mM MgATP for 25 min at 37 °C (during which hydrolysis rates were linear). Following the addition of 150 µl of stop (12% SDS, 1% ammonium molybdate in 1 M HCl) and 150 µl of stabilizing (2% (w/v) each of Na metarsenite, Na citrate, and acetic acid) solutions and a 10-min incubation at 37 °C, the absorbance was measured at 750 nm. Basal ATPase activity was measured as nmol of Pi liberated per min per mg of protein. Assays carried out in the presence of verapamil or orthovanadate were expressed as the percentage change in basal ATPase activity (mean ± S.E.) from at least five independent P-gp preparations. Background phosphate levels were measured by incubating samples in the absence of drug at 4 °C and were subtracted from all measurements.
[3H]Vinblastine BindingThe equilibrium binding of [3H]vinblastine to P-gp was determined using a rapid filtration assay based on published methods (14). Saturation isotherms were obtained by incubating 5-120 nM [3H]vinblastine with 20 µg of CHrB30 membranes or 1 µg of reconstituted P-gp in a total volume of 200 µl at 25 °C for 2 h. Samples were filtered through a combination of Whatman GF/F fiber and 0.1-µm nitrocellulose filters, and radioactivity retained was determined by liquid scintillation counting. Nonspecific binding of [3H]vinblastine binding to membranes and liposomes was determined in the presence of 3 µM unlabeled vinblastine. Data were expressed as pmol of [3H]vinblastine bound per mg of protein. The equation Bo = (Bmax × F)/(Kd + F) was fitted to saturation isotherms by nonlinear regression analysis using Kaleidograph (Abelbeck Software). Bo is the amount of [3H]vinblastine bound, Bmax the density of binding sites, Kd the apparent dissociation constant, and F the concentration of free [3H]vinblastine.
Photoaffinity Labeling of P-gpPhotoaffinity labeling of P-gp (1-2 µg of protein) with the 1,4-dihydropyridine [3H]azidopine (45 nM) was determined by fluorography of SDS-polyacrylamide gels as described previously (15). Vinblastine (1-500 µM, as indicated) was used to inhibit [3H]azidopine labeling.
Lectin-Gold LabelingLentil lectin agglutinin from
Lens culinaris (Sigma), specific for -mannose residues,
binds to glycosylated P-gp in the presence of divalent cations (16).
Detergent-solubilized P-gp and gold-labeled lectin (5 nm) were mixed
and incubated at a molar ratio of 1:1 at 4 °C overnight with
constant agitation in buffer (10 mM Tris-Cl, pH 7.5, 25 mM MgSO4). Specimen preparation for electron
microscopy was as described below.
Copper grids (400 mesh/inch; Agar Scientific Ltd., Stansted, United Kingdom) were coated with a collodion/carbon film. Specimens were adsorbed to freshly glow-discharged grids (to render them hydrophilic) for 30 s, washed with filtered water for 30 s, and negatively stained with uranyl acetate (4% w/v in water; Electron Microscopy Laboratories Ltd., Reading, UK) or sodium phosphotungstate (17) for 1 min. Electron micrographs were recorded at calibrated magnification using Phillips EM 301, Hitachi H-600, or Phillips CM 10 electron microscopes at 100 kiloelectron volts. Low dose conditions were achieved by focusing on an area of the carbon film adjacent to the area of interest to minimize irradiation damage to the specimen. The micrographs were digitized using a rotating drum microdensitometer (Joyce-Loebl) at 25-µm increments corresponding to pixel sizes in the range of 5-7 Å at the specimen level (depending on the primary magnification).
Image Analysis Using SPIDERSingle particle averaging was undertaken with the SPIDER software package on micro-VAX and Indigo-Silicon Graphics work stations (18, 19). The images were initially band pass-filtered (20). Particles were interactively selected using the program WEB padded to 64 × 64 pixels, and the densities were normalized (21). Padded images were masked with a circular mask, and a Gaussian falloff filter was applied with a half-width of 1.0 pixel. Particles were rotationally aligned by an autocorrelation function-based method (20) and translationally aligned by cross-correlation using a reference particle (22). Initially, the first 20 particles were aligned with respect to a reference particle, and the best aligned particles were used to calculate a new reference, which was then used to align the rest of the particles. An average image was calculated by summing the aligned particles. The images were then subjected to correspondence analysis (23) and classification (24), and the alignment procedure was repeated using the class averages as references. For resolution assessment, two independently derived averages were compared in Fourier space. The differential phase residual was computed and plotted as a function of the ring radius, and the resolution calculated when the 45° limit was reached. The Fourier ring correlation (25) was also used for resolution estimation.
Three-dimensional ReconstructionImages were taken in pairs at 100 kiloelectron volts, at an optimized level of defocus and under low dose conditions. The first image was taken with the specimen tilted by 45°, whereas the second was taken without tilt with the distances between the molecules optimized for minimal overlap during tilting. When the specimen is tilted, the particles adsorbing to the grid in a preferred orientation present a conical range of projections from which an averaged particle can be reconstructed in three dimensions. Particle images were selected simultaneously from 0 and 45° micrographs displayed side by side on a Silicon Graphics work station, using an interactive particle selection program that established the geometric relationship (in terms of rotation, translation, direction of tilt axis, and tilt angle) between the two micrographs. 400 particles from two electron micrograph pairs were selected (26) using the SPIDER image-processing system (23). The images were contrast-normalized and pretreated by band pass filtering to suppress the very low and high spatial frequencies (noise). The frequency limits were coupled to the size of the molecule (low frequency) and expected resolution (high frequency). A reference-free alignment algorithm was used, enabling a comparison of projection maps derived from reference-based and reference-free alignments (27). Untilted particles were classified (24) by hierarchical ascendant classification with complete linkage as a merging criterion. Construction of a linkage tree or dendrogram allowed partitioning of the data set into appropriate groups, based on group size and the "within-group resolution." In addition, an eigenvector plot was constructed according to the Diday method of classification (24). This plot shows the variations of the P-gp molecules in factor space, where the axes represent principal components of the variational pattern. Clusters were identified, and homogeneous subsets of images were then isolated.
For each group of particles (averages I-III) derived from hierarchical
ascendant classification, the corresponding tilt images (after
centering) were used for separate reconstructions according to the
random conical geometry. Reconstruction was undertaken by an iterative
three-dimensional reconstruction method based on a 2
minimization constraint (27). This method reduces the effects of
missing angular range on the reconstruction. Phase residual calculations were used for the cross-resolution assessment, and resulting reconstructions were low pass-filtered according to the
resolution assessment. For viewing the results, a surface representation was used on a work station using WEB in the SPIDER package.
Crystallographic image processing of small, two-dimensional crystalline arrays of P-gp was carried out using the program CRISP and Trimerge (Calidris Ltd., Stockholm, Sweden) (28). Images of micrographs (×86,400 magnification) were digitized at 64 µm/pixel, giving 7.4 Å/pixel at the specimen level. Crystalline arrays were displayed, and the best ordered areas were judged by the sharpness and intensity of the first order reflections in the calculated Fourier transforms. The best areas were then used for lattice refinement, extraction of amplitudes and phases, and plane group assignment. Structure factors from 10 separate crystalline areas were merged after phase origin refinement, and averaged phases and amplitudes were extracted for each spot. Since the plane group contained 1 asymmetric unit, the merging process required the operator to identify which of the two possible orientations of each crystalline area to apply. This decision was made by comparing the interimage phase residuals for the two possible orientations (rotated by 0 or 180° with respect to the first crystalline area used in the merging procedure); in each case the orientation with the lowest interimage phase residual was applied. The final averaged structure factors were used to calculate an averaged projection map of the unit cell. The averaged interimage phase residual was 31° (a residual of 90° would be expected for completely random data).
Chinese
hamster ovary CHrB30 cells have been selected for high
level drug resistance and overexpress P-gp to about 3% of total membrane protein. P-gp was solubilized from CHrB30
membranes using the nonionic detergent dodecyl maltoside and purified
to >95% by anion exchange and hydroxyapatite chromatography (see
"Experimental Procedures"). Fig. 1A shows
a silver-stained SDS-polyacrylamide gel of the starting material
(CHrB30 membranes) and purified P-gp. Purified P-gp was
reconstituted into liposomes of asolectin by gel filtration
chromatography. The efficiency of reconstitution was assessed by
co-migration of P-gp with tracer lipids during sucrose density gradient
centrifugation. P-gp did not reconstitute efficiently at lipid:protein
ratios of <10:1 (w/w). However, efficient reconstitution was obtained at higher lipid:protein ratios. The number of protein molecules reconstituted per unit area of liposome membrane observed by electron microscopy was as expected, given the relative amount of lipid and
protein used (see below).
P-gp activity was assayed by measuring drug-stimulated ATPase activity and substrate binding (Fig. 1, B and C). The basal ATPase activity of purified, reconstituted P-gp (800 ± 48 nmol/min/mg; n = 4) was significantly greater than that measured for CHrB30 membranes (377 ± 129 nmol/min/mg; n = 9) and compared favorably with the activity reported for P-gp purified from CHrB30 membranes by immunoaffinity chromatography (29). ATPase activity was stimulated by verapamil (EC50, 1.9 ± 0.3 µM) and inhibited by orthovanadate (EC50, 2.4 ± 0.7 µM), with half-maximal concentrations indistinguishable from those determined for P-gp in native membranes (Fig. 1B).2 Thus, coupling between substrate binding and ATPase activity was unaltered following purification and reconstitution. Purified, reconstituted P-gp retained high affinity [3H]vinblastine binding (Kd, 46 ± 10 nM; n = 4), similar to that measured for P-gp in native membranes (37 ± 5 nM; n = 3) (Table I). The specific binding capacity of purified P-gp for [3H]vinblastine (793 ± 108 pmol/mg) increased during purification (Table I). Azidopine, like other 1,4-dihydropyridines, is believed to bind at an allosteric site on P-gp (14). Concentrations of 1-5 µM vinblastine were required to displace 50% [3H]azidopine photolabeling of purified P-gp (Fig. 1C), indistinguishable from the concentrations required to displace [3H] azidopine binding from native membranes (15). Thus, the binding sites for azidopine and vinblastine remain coupled in a negative allosteric fashion in the purified P-gp. In conclusion, the activities of purified and reconstituted P-gp were not significantly different from those of P-gp in native membranes.
|
Electron microscopy and single particle averaging (30) allow multiple images of a macromolecule in a characteristic orientation to be translationally and rotationally aligned using an iterative procedure of cross-correlation and autocorrelation and then averaged to produce a refined image (25). Single particle analysis was initially carried out on reconstituted P-gp because, compared with solubilized protein, the lipid bilayer confines all the particles in the z axis (i.e. the axis perpendicular to the membrane plane) such that they can only exhibit rotational freedom within the x, y plane, limiting the potential orientations that the molecules can adopt.
Proteoliposomes were negatively stained with uranyl acetate and
examined by electron microscopy. The lipid vesicles were relatively uniform in size, between 50 and 200 nm in diameter (Fig.
2A). Small, uniform, 10-nm-diameter particles
with a heavily stained central region were observed in these vesicles
(Fig. 2A). The particles were also observed after staining
with sodium phosphotungstate, pH 7.0 (data not shown). The contrast for
reconstituted protein was low compared with detergent-solubilized
protein (see below), as expected for a membrane-embedded protein
protruding only a small distance from the bilayer. A montage of several
typical particles at a higher magnification and with contrast maximized is shown in Fig. 2A. These particles were not observed in
lipid vesicles in the absence of reconstituted P-gp. The area occupied by the particles at a 10:1 lipid:protein ratio, as a proportion of
total membrane surface area, was 1:23, in good agreement with the
expected value calculated from the partial specific volumes of lipid
and protein and the greater "depth" of the protein (8 nm; see
below) compared with the 4-nm lipid bilayer.
Protein molecules reconstituted into lipids normally present a uniform, face-on view because of vesicle flattening on the support film of the grid, and because uranyl acetate preferentially stains the membrane plane most closely associated with the support film (17, 31). The signal:noise ratio was enhanced by averaging images of many particles with similar projections relative to the electron beam (30). The images of individual particles were first aligned and then classified into groups displaying similar projections by correspondence analysis (21, 23). Then, within each class, the particles were translationally and rotationally aligned with respect to a reference particle selected from the group. Such methods have significantly enhanced the structural information obtained for large macromolecules such as the ribosome (25) and chaperonins (32).
Classification of the reconstituted P-gp showed that >70% of the
molecules represented the same view. This is consistent with previous
reports that, following reconstitution, >80% of P-gp is in the same
orientation (16). Images of 25 individual particles with a high
signal:noise ratio were aligned with respect to reference particles
(18) and then averaged to produce an initial projection map of the
protein (Fig. 3C). Further averaging with 135 particles generated the projection map shown in Fig. 3F.
Since the molecules are fixed in the dimension parallel to the membrane
plane (33), it is possible to use a relatively small number of
molecules for the final average (25). The general structures of these
two projections were very similar, although some detail was lost by averaging 135 particles due to increased variability in staining and
imperfect rotational alignment of those particles with poorer signal:noise ratios. In this projection the protein is toroidal, with a
ring of protein exhibiting hexagonal symmetry surrounding a 5-nm
central pore. The intense staining of the pore with both uranyl acetate
and sodium phosphotungstate implies it is aqueous. P-gp has a single
glycosylation site at the extracellular face of the membrane, and
lectin-gold labeling showed that this view corresponds to the face of
the protein exposed extracellularly (see below).
Electron Microscopy and Image Analysis of Single Particles of Detergent-solubilized P-gp
To obtain projections of other orientations of P-gp, detergent-solubilized protein was studied. Fig. 2B shows that the detergent-solubilized protein consists of an essentially homogeneous collection of 10-12-nm-diameter particles. The sizes of the solubilized and reconstituted particles were similar, showing that their oligomeric state is the same. As expected, and in contrast to the reconstituted protein, solubilized P-gp showed more than one orientation with respect to the grid support film. However, the particles were not randomly orientated because of differential association of different faces of the protein with the electrostatically charged grid surface (21). Classification of 621 particles by correspondence analysis (21) identified three distinct projections, which represent distinct orientations of the protein with respect to the electron beam. A representative eigenvector plot is shown (for the untilted data used in the three-dimensional reconstruction, see below). The three projection maps corresponding to these three orientations are shown in Fig. 3, A, D, and E. Each final projection map was generated from at least three rounds of iterative refinement. Similar projection maps were obtained from multiple independent preparations of P-gp.
One projection (Fig. 3D) closely resembled that of the reconstituted protein (Fig. 3C) and therefore represented the extracellular face of the protein. The detergent-solubilized protein had slightly larger dimensions (12 nm) in this projection than the reconstituted protein, probably reflecting an annulus of lipid or detergent molecules surrounding the protein (16, 34). The second projection (Fig. 3E) was also circular and 10-12 nm in diameter. However, this projection was distinct from the extracellular face-on views shown in Fig. 3, C, D, and F; there was no central pore, and two 3-nm lobes could be identified. These lobes are an appropriate size for the 200-amino acid nucleotide binding domains (5, 6). This projection was shown to correspond to a face-on view from the cytoplasmic face of the membrane by three-dimensional reconstruction (see below). The third projection (Fig. 3A) was asymmetric in shape with a strongly stain-excluding, three-lobed structure across the center of the protein. This is indicative of a hydrophobic region normally embedded in the membrane and suggested that this projection was likely to be a side-on view of P-gp. Two 3-nm domains (Fig. 3A, arrows) extend from one side of this central structure. Studies on crystalline arrays of P-gp and three-dimensional reconstructions confirmed that this view represents a side-on view of the molecule and that the two 3-nm domains are on the intracellular face of the molecule (see below). In this side-on projection, P-gp is compact, about 8 nm in depth, which is about twice the depth of a lipid membrane bilayer (4 nm). The three-lobed appearance of the core of the protein in this side-on projection (Fig. 3A) is consistent with a side-on view of a protein with the hexagonal symmetry seen in the face-on projections (Figs. 3, C and D, and 4F).
Three-dimensional Reconstruction of Detergent-solubilized P-gpUsing single particle averaging it is possible to calculate a three-dimensional structure based on a random conical technique (26) and reference-free alignment algorithm (27). A specimen field containing particles in the preferred orientation was imaged with the specimen tilted to 45° and with the specimen in the untilted position. Particle projections appearing in the tilted image form a random conical projection set, since the particles assume random orientations with respect to an axis perpendicular to the specimen plane (27, 35-37). One of the major advantages of this method is that only one exposure of the specimen area is necessary, thus minimizing radiation damage to the molecule. A second exposure of the same specimen area is taken without tilt, but this is not used for the reconstruction but for measurement of the in-plane rotations and for the azimuthal placement of the corresponding tilted specimen projection on the cone. An iterative three-dimensional reconstruction method was then used to remove artifacts caused by missing angular information (27).
The micrographs shown in Fig. 2B represent a typical field
of the untilted molecules used for three-dimensional reconstruction, except that the protein density of the sample used for this analysis was lower than that shown. After alignment, the untilted images were
subjected to correspondence analysis and classification (21). The
images fell into three major classes (designated I-III) by hierarchical ascendant classification, representing different preferred
orientations of P-gp molecules on the grid. A dendrogram was calculated
(Fig. 4A) showing the classes generated at a
cutoff level of 0.1. The decision for cutting the dendrogram at this threshold was based on the demonstration of distinct orientations for
each of the three major classes and a good within-group resolution (1/25 Å1). The numbers of particles in classes I-III
were 171, 94, and 47, respectively. After averaging (Fig.
4C), each class resembled one or more of the projection map
averages obtained from single particle analysis (Fig. 3). The average
obtained for class I showed an extracellular face-on character slightly
tilted in the plane of the membrane, with a hexagonal shape and a 5-nm
central pore (as in Fig. 3C); the average obtained for class
II had side-on characteristics, with a central stain-excluding central
region (characteristic of the single particle projections shown in Fig. 3, A and B); the average obtained for class III
also resembled an extracellular face-on projection but tilted at a
different angle compared with class I particles.
Definition of particle classes for
three-dimensional analysis. A, the linkage tree obtained
from hierarchical ascendant classification using
complete linkage as a merging criterion. The cutting
level used was 0.1, which generated a suitable within group resolution
of the major classes (1/25 Å1). B,
eigenvector plot of factor 1 versus factor 2 showing three major clusters (circled). C, averages of the
three most populated classes determined by hierarchical ascendant
classification. Class I particles were used for the three-dimensional
reconstruction and represent a partial (slightly tilted) face-on view
of the molecules. Bar, 5.3 nm.
The eigenvector plot (Fig. 4B) shows each image as a symbol representative of the cluster to which it belongs, using an alternative method of classification (the Diday method). The first two factors were identified as the most important for representing the image set; thus the main division of the data was shown in a display of factor 1 versus factor 2. The symbols (1-9 and A-Z) follow the ranking of the clusters, with cluster 1 being the most abundant. By averaging over images in clusters 1-3, the same motifs were again obtained, and these three clusters corresponded closely to classes I-III described above. The demonstration that two alternative classification methods (Diday versus hierarchical ascendant classification) generated similar averages implied that stable orientations were assumed by the particles and that the final partition in our classification scheme was largely correct.
As the P-gp molecules prepared on a single carbon layer could be
classified into three distinct classes of views, three-dimensional reconstructions were performed, separately, on each of these classes. Fig. 5 shows a computer-generated montage of the
three-dimensional structure of P-gp using surface rendering to give an
impression of the overall shape of the molecule. This surface was
calculated from the major class of particle (class I). This
three-dimensional model represents the whole molecule, because
information from the opposite side of the protein is derived from the
thinly distributed stain, visualized by tilting the specimen support
film. Merging the random conical data sets obtained from each of the
differently oriented classes of particle was not necessary to obtain an
initial structure, because classes II and III contained significantly fewer particles than class I, giving a poorer resolution. Each particle
class, however, yielded essentially the same three-dimensional surface.
Similar results have been described by Carazo et al. (37)
for the 50 S ribosomal subunit. The estimated resolution of this
structure was 4.7 nm as determined by phase residual analysis of the
three-dimensional volumes.
The initial image of the displayed molecule (Fig. 5A) corresponds to a view from the extracellular face of the membrane. The displayed molecule has been rotated around a horizontal axis, with the direction of rotation moving the top of the molecule in the panel away from the observer. The rotation sequence in the montage finishes after a 192° rotation. Small satellite features disconnected from the main body of the molecule are probably due to residual noise in the three-dimensional map and have been ignored in the discussion of the structure. The threshold level used for this surface-rendering display was chosen so that this peripheral noise was virtually eliminated. At this threshold, the overall dimensions of the three-dimensional representation correlated well with the dimensions of the molecule derived from contour maps of projections from untilted particles (Fig. 3).
Fig. 5A is equivalent to the two-dimensional projection maps
shown in Fig. 3, C, D, and F (see Fig.
3C in particular, with its elongated hexagonal form) and is
interpreted to represent a view perpendicular to the extracellular face
of the membrane (see above). This view is shown at higher magnification
in Fig. 6A. As expected, the
three-dimensional structure revealed domains that were unresolved in
the two-dimensional projection maps because these features were
superimposed in the projected structures. The ring of protein
surrounding the central pore consists of two thumb-shaped domains, each
of which consists of three partially resolved subdomains. The tip of
each "thumb" rests on the base of the other one, giving this part
of the molecule a twisted but relatively symmetrical appearance, with
an approximate 2-fold axis of symmetry about the center of the pore.
Together these thumb-shaped structures correspond to the toroidal
structure seen for reconstituted protein from the external face of the
membrane (Fig. 3C). The pore is approximately 2.5 nm in
diameter (Fig. 5), although when measured at the outer surface (Fig. 3)
it is about 5 nm. Thus, the chamber within the membrane is conical, narrowing toward the cytoplasmic face of the membrane. The
three-dimensional reconstruction shows the conical nature of this
chamber.
Located beneath the intertwined thumbs in the top left panel, and partially obscured by them, are two further domains that are also symmetrically related. These two domains are more apparent in side-on orientation and are approximately 3 nm in diameter (Fig. 5, straight arrows, shown at greater magnification in Fig. 6B). In three-dimensional reconstruction these domains can be seen to be positioned on the opposite (intracellular) side of the molecule to the thumbs and correspond to the 3-nm lobes seen in the intracellular face-on projection (Fig. 3E). They were not seen in the extracellular face-on projection of the reconstituted protein (Fig. 3C), as they would have been obscured by the lipid bilayer. Rotation of the molecule through approximately 90° shows that these two intracellular domains extend outward, giving the side view of the protein a truncated conical shape. They do not project far into the cytoplasm but are intimately associated with the rest of the molecule such that P-gp is relatively compact with a depth of about 7.5-8.0 nm in the plane of the membrane. Each intracellular domain appears to be more contiguous with one thumb than the other, enhancing the apparent double helical packing of the protein. The left domain is slightly larger than the right domain, with an additional mass extending downward. Although it is not possible to rule out, unambiguously, the possibility that this additional mass may be due to residual noise in the map, this seems unlikely, as a similar asymmetric appearance was also noted from the side view projection maps (Fig. 3, A and B). Thus, the two intracellular domains appear to exhibit structural differences within the molecule. It is likely that these intracellular domains correspond to the nucleotide binding domains (see "Discussion").
A further major feature of P-gp is an elongated cavity formed between the left intracellular domain and the thumb lying closest to the observer in the side-view (Figs. 5C, curved arrow, and 6B, straight arrow). This opening does not have a symmetry-related equivalent on the other side of the molecule. This asymmetry is also seen as a "gap" in the toroidal representation of P-gp seen from the extracellular projection of the reconstituted protein (Fig. 3C). This opening is located within the region of protein predicted to be embedded in the lipid bilayer and could provide access between the lipid phase and the aqueous chamber within the protein core (see "Discussion").
Analysis of Small Two-dimensionally Ordered Arrays of P-gpSmall, single layers of epitaxial two-dimensional crystals
of P-gp formed spontaneously when the electron microscopic grid support
film was incubated with concentrated (0.3 mg/ml) solutions of
detergent-solubilized P-gp. These are shown in Fig. 2B
(boxes). The arrays are small and superimposed by many
randomly oriented (nonordered) protein molecules that are distributed
throughout the grid, making the arrays hard to visualize. Image
processing of these arrays (Fig. 7) was therefore
required to average the repeat units and to average out the randomly
orientated protein. The detection of small crystalline arrays
demonstrates the structural homogeneity of the protein prepared by this
purification methodology. The crystal packing was similar to that
observed for some three-dimensional membrane protein crystals in which
lattice contacts occur between hydrophilic extracellular and
intracellular loop regions, presumably because the bulky annulus of
disordered detergent molecules around the transmembrane regions
prevents lattice contacts between hydrophobic regions (38). Fourier
transforms of selected crystalline areas showed visible diffraction
with a signal:noise ratio >5, sufficient to measure the lattice
parameters and to apply image processing via Fourier filtering
(i.e. extraction of structural information from the
surrounding noise). Merging the data from 10 separate small crystalline
areas significantly enhanced the signal:noise ratio of the resulting
averaged projection map, giving structural data up to the fifth
diffraction order (~3 nm resolution) (Fig. 7) with an average
(amplitude-weighted) interimage phase residual of 31°. The unit cell
of these crystals had a symmetry with the plane group containing 1 asymmetric unit, with a = 14.2 nm, b = 18.5 nm, and = 91.6°. These dimensions are slightly larger than
those predicted by single-particle analysis. This could be due to the
direction of packing in the lattice producing a longer b
axis than expected. The projection map calculated from these arrays
(Fig. 3B) showed a strong resemblance to one of the
projections (the side-on projection) obtained by single-particle
averaging (Fig. 3A). It should be noted that the map
generated by single-particle alignment was at a slightly higher
resolution (2.5 nm) than that generated by Fourier analysis. A poorly
staining, three-lobed central structure was observed, consistent with a
side-on view of a molecule with hexagonal symmetry in its face-on view
(Fig. 3, C, D, and F). Two 2-3-nm-diameter lobes
protrude from one side of the molecule (Fig. 3B, arrows),
which correspond to the intracellular lobes (putative NBDs) obtained by
three-dimensional reconstruction from single particle images (see
above).
Lectin-Gold Labeling of P-gp
P-gp is glycosylated on a single
extracellular hydrophilic loop (1). Lectin-gold labeling was used to
evaluate the orientation and oligomeric state of P-gp (Fig.
8). It should be noted that this preparation was
slightly less pure (75% compared with >95%) than those used for
image analysis, possibly explaining why not all the molecules are
labeled. Aggregation was also observed, probably due to the overnight
incubation. The lectin-gold consistently labeled the face of the
protein corresponding to the projection shown in Fig. 3, C,
D, and F, showing that these projections correspond to
the face of P-gp exposed at the extracellular face of the membrane (as
glycosylation only occurs on an extracellular site). No more than one
gold particle was ever associated with a nonclustered P-gp particle,
even when the majority of particles were labeled, demonstrating that
lectin-gold was not limiting. Thus, active P-gp appears to be
monomeric. However, these data do not formally exclude the possibility
that P-gp is dimeric and that dimerization sterically occludes one of
the glycosylation sites such that the dimer can only be labeled by a
single lectin-gold particle.
The absence of structural data for any ABC transporter has limited our understanding of the mechanisms of active transport. In this study we have used electron microscopy to generate an initial structure (to 2.5 nm resolution) of the multidrug resistance P-glycoprotein. Three separate approaches were used that gave consistent results and, together, enabled the orientations of images with respect to the membrane to be ascertained: two-dimensional projections from single particle imaging of lipid-reconstituted protein; two-dimensional and three-dimensional reconstructions from single particle image analysis of detergent-solubilized P-gp; and Fourier transform projection maps of small crystalline arrays of P-gp. These are the first structural data for any ABC transporter.
P-gp was purified from membranes of cells selected for high level drug resistance and retained activity following purification as assessed by drug binding and drug-stimulated ATPase activity. The P-gp particles observed by electron microscopy were of similar dimensions whether detergent-solubilized or reconstituted into liposomes. Lectin-gold labeling indicated that purified P-gp is monomeric. The volume of the structure determined here is also consistent with a monomer (see below). As purified P-gp retains activity, this suggests that P-gp can function in the monomeric state. Other recent, biochemical studies have also suggested P-gp can function as a monomer (39), and another ABC protein, cystic fibrosis transmembrane conductance regulator, also appears to be monomeric (40). Whether P-gp is monomeric in native membranes or forms oligomeric aggregates is not known. It has previously been suggested that P-gp may be oligomeric, based on radiation inactivation and cross-linking of P-gp overexpressed in native membranes (41-43).
From the data presented here, an initial structural model for P-gp has
been derived (Fig. 6), which is entirely consistent with available
biochemical and genetic data. In overall shape, P-gp approximates a
cylinder of about 10 nm in diameter with a maximum height (in the plane
of the membrane) of about 8 nm. This compares with a depth of the lipid
bilayer of about 4 nm, suggesting that about one-half of the molecule
is within the membrane. Viewed from the extracellular surface of the
membrane (identified by reconstitution into lipids and by lectin-gold
labeling), P-gp is toroidal with a large central pore of about 5 nm in
diameter. The ring of protein surrounding the central pore is roughly
hexagonal, consisting of two thumbs, each with three lobes (seen most
clearly in Figs. 3, C and D, and 6A).
The three-domain appearance in side-on views of the membrane region of
P-gp (Fig. 3, A and B) is also consistent with an
overall 6-fold symmetry. Biochemical data have shown that P-gp consists
of 12 membrane-spanning segments (3, 4), each predicted to exist as an
-helix. These 12 membrane-spanning segments are separated by six
extracellular hydrophilic loops, one of which is glycosylated. The
internally repeated amino acid sequences of the two transmembrane
domains predict a 2-fold pseudosymmetry. It is, therefore, reasonable
to speculate that the two thumbs represent the two transmembrane
domains and that the six lobes (three per thumb) reflect the six
extracellular loops connecting pairs of membrane-spanning segments
(putative
-helices).
The aqueous pore open at the extracellular face of the membrane is much larger in diameter (~5 nm) than is required for the passage of known P-gp substrates. Such a large pore, if open across the membrane, would destroy the permeability barrier. However, the pore is closed at the cytoplasmic face of the membrane. Thus, P-gp forms a large aqueous chamber within the membrane, open to the extracellular milieu. In addition, an opening to the lipid phase, within the plane of the membrane, is also apparent. This opening is consistent with the "flippase" model for P-gp and data showing that substrates can gain access to the pore-translocation pathway from the lipid phase (44-46).
P-gp has two 3-nm lobes at the cytoplasmic face of the membrane. The two NBDs of P-gp are the only substantial portion of P-gp not embedded in the membrane and are primarily cytoplasmic (4, 5). The 3-nm lobes are of an appropriate size for the 200 amino acid NBDs. It is, therefore, likely that these lobes correspond to the NBDs. Each lobe (putative NBD) is more closely associated with one of the transmembrane thumbs than with the other. The two lobes (putative NBDs) appear asymmetrically organized within the P-gp molecule. It is known that P-gp undergoes conformational changes in the presence of ATP and substrate (47). The present structural data were obtained in the absence of ATP; it will be interesting to ascertain whether the conformation or asymmetry of these lobes (putative NBDs) is altered on ATP binding and/or hydrolysis.
The surface area occupied by P-gp in the membrane (the 12 predicted
membrane-spanning -helices per monomer) is approximately 60 nm2. For comparison, cytochrome oxidase (22 membrane-spanning helices) occupies an area of about 54 nm2
(48), the bacterial photoreaction center (11 membrane-spanning helices)
an area of 32 nm2 (49), and light-harvesting complex 2 (18 helices) an area of about 49 nm2 (50). Thus, if P-gp is
monomeric, the transmembrane helices of P-gp must be relatively loosely
packed. The mass of a solid 10 × 8-nm cylinder (the overall
dimensions of P-gp) can be calculated as about 540 kDa, assuming 0.7 cm3 g
1 as a partial specific volume for the
protein (51). Although protein-packing and volume calculations are
notoriously variable, this is closer to the mass of a dimer, rather
than that of the 170-kDa P-gp monomer. However, the structural data
show that the protein is, indeed, loosely packed, and the central
chamber and spaces between intracellular lobes (putative NBDs) occupy a
significant proportion of the volume of P-gp. When these factors are
taken into account, the structure is not inconsistent with a 170-kDa monomer. Interestingly, hexameric annexin has approximately the same
overall dimensions as P-gp (a 10 × 7-nm cylinder with a large central pore) and is of similar molecular mass, 210 kDa (52).
The aqueous chamber within the membrane is closed at the cytoplasmic face of the membrane. This closure is presumably achieved by the two intracellular lobes (putative NBDs) and the hydrophilic cytoplasmic loops that separate the 12 membrane-spanning segments. This may provide an explanation for the finding that the NBDs of certain ABC transporters are accessible from the outside of the cell yet do not contain segments that would normally be expected to span the bilayer (53, 54); the transmembrane pore is sufficiently large to allow reagents to gain access to the NBDs at the intracellular face of the membrane. Most kinetic models of transport predict the alternate exposure of a substrate binding to the two sides of the membrane. As the energy required to reorientate a binding site from one face of the membrane to the other would be large, it has generally been assumed that any conformational change would be relatively localized within the membrane. The structure of P-gp suggests that this occurs at the inner face of the membrane. This is consistent with data that show that the substrate binding site of P-gp is at the cytoplasmic face of the membrane: it is accessible from the inner face of the membrane3; substrates can be cross-linked to regions of the protein predicted to be at the cytoplasmic face of the membrane (55); and mutations altering amino acids at the cytoplasmic face of the membrane can influence substrate selectivity (9, 56).
P-gp is a member of the ABC superfamily of transporters, and it is not unreasonable to suppose that the general architecture of other ABC transporters may be similar to that of P-gp. Some ABC transporters handle very large substrates (e.g. polypeptides or polysaccharides), whereas others handle rather small substrates (e.g. inorganic ions). A common architecture with a large pore could readily be adapted to accommodate different sized substrates with minor changes to the "gate" at the cytoplasmic face of the membrane. The availability of structural data for P-gp is necessary for directing future experiments aimed at understanding the molecular mechanisms by which P-gp and other ABC transporters operate.
We are extremely grateful to Dr. J. Frank (Wadsworth Center, NY) for enabling M. F. R. to visit his laboratory to undertake three-dimensional reconstruction techniques and Dr. Pawel Penzek and Dr. Ramani Kharidehal for assistance. We also thank Dr. Andreas Holzenburg (University of Leeds, Leeds, UK) for reading the manuscript, Dr. Richard Henderson and Dr. Nikolaus Grigorieff (Medical Research Council Laboratories, Cambridge, UK) for help with data analysis, and Kenny Linton, Jim Mallet, Paul McPhie, Matthew Grimshaw, Candida Nastrucci, and Gail Begley for discussions.