From the Department of Biomolecular Sciences,
University of Manchester Institute of Science and Technology,
Manchester M60 1QD, United Kingdom, the
Department of Clinical
Laboratory Sciences, University of Oxford, John Radcliffe Hospital,
Oxford OX3 9DU, United Kingdom, and the ** Medical Research
Council Clinical Sciences Centre, Faculty of Medicine, Imperial
College, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN,
United Kingdom
Received for publication, November 19, 2002
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ABSTRACT |
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P-glycoprotein is an ATP-binding cassette
transporter that is associated with multidrug resistance and the
failure of chemotherapy in human patients. We have previously
shown, based on two-dimensional projection maps, that P-glycoprotein
undergoes conformational changes upon binding of nucleotide to the
intracellular nucleotide binding domains. Here we present the
three-dimensional structures of P-glycoprotein in the presence and
absence of nucleotide, at a resolution limit of ~2 nm, determined by
electron crystallography of negatively stained crystals. The data
reveal a major reorganization of the transmembrane domains throughout
the entire depth of the membrane upon binding of nucleotide. In the
absence of nucleotide, the two transmembrane domains form a single
barrel 5-6 nm in diameter and about 5 nm deep with a central pore that
is open to the extracellular surface and spans much of the membrane
depth. Upon binding nucleotide, the transmembrane domains reorganize
into three compact domains that are each 2-3 nm in diameter and 5-6
nm deep. This reorganization opens the central pore along its
length in a manner that could allow access of hydrophobic drugs
(transport substrates) directly from the lipid bilayer to the central
pore of the transporter.
ATP Binding Cassette
(ABC)1 transporters are an
extended family of membrane proteins defined by a highly conserved
domain, the ATP binding cassette (1); they mediate the
ATP-dependent transport of a wide variety of compounds
across cellular membranes (2, 3). The core ABC transporter consists of
two transmembrane domains (TMDs) and two nucleotide binding domains
(NBDs). The NBDs are peripherally located at the cytoplasmic face of
the membrane, bind ATP, and couple ATP hydrolysis to the transport
process. All NBDs whose structures have been determined have very
similar tertiary folds (4-8). The TMDs bind the transported substrate and form the pathway through which it crosses the membrane. In contrast
to the NBDs, the TMDs of different ABC transporters share little
primary sequence similarity, except between closely related members of
a subfamily; this may be because of the variety of substrates
transported by different ABC proteins. Little is known about the
structures of the TMDs of ABC transporters or how the binding/hydrolysis of ATP by the NBDs is coupled to transmembrane transport of solute. Hydrophobicity plots typically predict six transmembrane P-glycoprotein (P-gp) is a mammalian ABC transporter that
pumps hydrophobic drugs across the cell membrane and can confer multidrug resistance on cells and tumors. P-gp is probably the best
characterized ABC transporter, and much is known about the ATP
hydrolytic cycle (12-14) and drug binding sites (15-18). There is a
body of biochemical evidence suggesting that the TMDs undergo conformational changes upon nucleotide binding, including changes in
epitope accessibility (19, 20), protease susceptibility (21, 22), drug
binding (17), fluorescence, and spectroscopic measurements (23-26). To
understand the mechanism of transport, these biochemical data need to
be linked to structural information. We have previously reported low to
medium resolution structures for P-gp determined by both single
particle image analysis (27) and by electron crystallography of
two-dimensional crystals (28). The two-dimensional projection
maps for P-gp trapped at different stages of the hydrolytic cycle
suggest substantial conformational changes at the extracellular face of
the TMDs upon binding the non-hydrolyzable ATP analogue
adenylyl-imidodiphosphate (AMP-PNP) and after vanadate-trapping in the
presence of ADP (ADP/Vi state) (28). We have now generated a
three-dimensional structure for P-gp in the presence of AMP-PNP and
compared this with the three-dimensional structure of P-gp in the
absence of nucleotide. The data show substantial conformational changes
throughout the TMDs of P-gp upon nucleotide binding, requiring
significant repacking of the transmembrane P-glycoprotein was purified from CHrB30 Chinese
hamster ovary cells selected for over-expression of P-gp (29).
Two-dimensional crystals were grown in the presence or absence of
nucleotide and negatively stained with uranyl acetate, as described
previously (28), using hanging-drop methods developed by Auer et
al. (30, 31). Where appropriate, 5 mM AMP-PNP, a
non-hydrolyzable analogue of ATP, was added directly to the
crystallization droplet. Electron microscopy was under low-dose
conditions. Images were digitized on a UMAX Power Look 3000 densitometer at 0.41 nm/pixel at the specimen level. Lattice unbending
and contrast transfer function correction were as described earlier
(28). Structure factors were merged with ORIGTILTD and averaged with
the program LLFILT to give interpolated structure factors along each
lattice line (Fig. 1), as previously described (32,33).
Determination of the correct orientation of each crystal relative to
the core data set was crucial. The crystals are in the p1
plane group, the lattice dimensions a and b are
similar, and the ab angle ( Crystallization of P-gp and Generation of Three-dimensional
Structure Maps--
Two-dimensional crystals of highly purified
P-glycoprotein in detergent were grown in the presence or absence of
AMP-PNP, a non-hydrolyzable analogue of ATP that is known to bind to
the NBDs at the same site as ATP. Three-dimensional structures were generated by electron crystallography (see "Experimental
Procedures"). Two-dimensional crystals formed more readily in the
presence of AMP-PNP, suggesting that this compound favored either the
nucleation or stability of the two-dimensional crystals. Crystal order
was also slightly better with AMP-PNP, as shown by comparison of the S.D. of the mean phases in most of the resolution ranges for
untilted data (Table I). Crystals grown
with and without AMP-PNP were similar in size (in excess of 1 micron
across), but the unit cell area was slightly smaller in the presence of
AMP-PNP (Table I). In both conditions, crystals had a p1
plane group, so that symmetry operations could not be used to judge the
quality of the structural data. Instead, crystal-to-crystal variation
and resolution limits for the data were assessed by analyzing the
deviations from the (vector sum) mean phase (Table I). These began to
increase around 2 nm resolution for the P-gp-AMP-PNP crystals, but even
at this limit phase errors were significantly lower than those expected for random data. Visual assessment of the scatter present in the three-dimensional data for the P-gp-AMP-PNP crystals was achieved by
plotting the structure factors of lattice lines along z* in reciprocal
space (Fig. 1), suggesting that the
three-dimensional structural data could be relied on to about 2 nm
resolution. At this limit, domains in P-gp were resolved, but secondary
structures such as transmembrane
In two-dimensional crystals of nucleotide-free-P-gp (nf-P-gp), the
molecules were packed such that they are slanted across each other
(Fig. 2, panel A, angled away
from the observer) with the long axis of each molecule oriented about
25-30o from the normal to the crystal plane. Such packing
would be disallowed in crystals formed by reconstitution in lipid
bilayers but can be accommodated in these crystals, which were grown in
the presence of detergent micelles. In contrast, in crystals of
P-gp-AMP-PNP the molecules were aligned with their long axis almost
exactly perpendicular to their two-dimensional crystal plane (Fig. 2). These packing differences, at least in part, explain the smaller unit
cell area of the P-gp-AMP-PNP crystals (Table I).
Comparison of the Three-dimensional Structures of P-gp in the
Presence or Absence of AMP-PNP--
The three-dimensional maps of
nf-P-gp and P-gp-AMP-PNP (Fig. 2) each comprise high and low density
regions (the high density region is closest to the observer in
panels A and B and at the top in panel
C). The high and low density regions were more pronounced for
nf-Pgp (Fig. 2, panel A). Because the high density region is
the side of the molecule in contact with the support film, the
differences in the two regions are likely to be because of the
well documented `differential staining' effect (36, 37) in which
better contrast, and hence higher apparent protein density, is observed
in the region closest to the support film. We have previously shown by
lectin-gold labeling (27, 28) that the surface of the P-gp molecule in
contact with the support film corresponds to that exposed at the
extracellular face of the membrane. Thus, the high-density region
corresponds to the TMDs, whereas the low density region corresponds to
the NBDs.
The three-dimensional structure of nf-P-gp is shown in panel
A of Fig. 2. The high density region, corresponding to the TMDs, resembles a barrel 5 to 6 nm in diameter and about 5 nm long. The
barrel surrounds a central pore, which appears to be open at the top
(equivalent to the extracellular face of the membrane) and closed at
the bottom (intracellular). The overall structure of the TMDs is very
similar to that determined previously by the entirely different method
of single particle image analysis (27). The three-dimensional netting
display used here gives a clear impression of the full
three-dimensional volume, allowing the identification of features in
the map not seen previously, in particular a density that protrudes
toward the central axis of the pore folding in from the bottom wall of
the barrel and a smaller density protruding into the pore from the
top of the barrel. After suitable rotation of the structure (see legend
to Fig. 3), a view directly down the
barrel was obtained (Fig. 3a, bottom panel). Note
that the walls of the barrel are roughly 1-1.5 nm thick, which roughly
equates to the diameter of a transmembrane
In contrast to the nf-P-gp structure, the high density (TMD) region of
P-gp-AMP-PNP consists of three clearly segregated domains (designated
A, B, and C in Fig. 2B).
Two of these domains are roughly equivalent in size and shape, with a
footprint of about 3 × 1.5 nm and a length perpendicular to the
crystal plane of about 4.5 nm. The third domain (C) has a
smaller footprint (about 2 nm diameter) but is somewhat longer
perpendicular to the plane of the membrane (about 6 nm). Domain C was a
useful reference point in the data merging procedure, because its
smaller footprint but higher density allowed it to be distinguished
from the other two domains in projection maps of individual crystals
(see Fig. 2, panel B). The three domains of P-gp-AMP-PNP
also enclose a central `pore.' However, unlike nf-P-gp the pore is
less obviously closed at the bottom (intracellular face of the
membrane) and, additionally, is open to the lipid phase along one side
with a gap appearing between two domains (Fig 2, panel B,
arrow). The opening up of this gap may explain why the
P-gp-AMP-PNP crystals are less affected by differential staining with
apparently better penetration of stain through the molecule (compare
Fig. 3, a and d, top panels).
The low density regions of each P-gp map correspond to the NBDs (28).
Because the NBDs are farther from the grid support film, they are less
contrasted by heavy atom stain and less well protected against the
damaging effects of the high vacuum and electron beam in the microscope
(36, 37). Thus, densities in this region are weak, especially for
nf-P-gp (Fig. 3a), and are therefore more difficult to
interpret than the densities for the TMDs (4, 8). The characterization
of the NBDs must await a three-dimensional structure for unstained
two-dimensional crystals of P-gp obtained by cryo-electron microscopy.
Comparison of P-gp with Bacterial ABC Transporters--
The
crystal structures of two bacterial ABC transporters, MsbA and BtuCD,
have recently been determined (9, 11). These structures differ
substantially from each other. We attempted to fit high-resolution
coordinates for the protein backbone of the TMDs from both bacterial
transporters to the high-density (TMD) region of nf-P-gp (the bacterial
ABC structures were determined in the absence of nucleotide ligand).
The global structure of BtuCD is similar to that of nf-P-gp (Fig. 3),
but the 20 transmembrane We have determined and compared the three-dimensional structures
of nf-P-gp and P-gp-AMP-PNP. Because of the way the crystals were grown
and stained, the NBDs were not clearly delineated. The TMDs of nf-Pgp
form a barrel-like structure surrounding a pore, open at the
extracellular face of the membrane and closed at the cytoplasmic face.
This structure was similar to that previously determined by us using
other approaches (27, 28), and recently by others at low resolution in
a lipid environment (38), although in this three-dimensional display
more detail could be seen. In particular, small densities protruding
into the pore could be seen. Their role in the transport process is unknown.
The new structural data show substantial reorganization of the TMDs of
P-gp upon binding nucleotide. We have previously reported such
structural changes (28), but because these were observed from
projections of the molecule the maps were restricted to two-dimensions. The three-dimensional structures presented here show that these conformational changes occur throughout the depth of the membrane and
must therefore involve repacking of the transmembrane Unlike the NBDs, the TMDs of different ABC transporters share little
sequence homology, except within very closely related sub-families. It
is therefore unclear whether the TMDs of different subfamilies of ABC
transporters are related to each other either evolutionarily or
structurally. The two high-resolution structures reported for bacterial
TMDs (9, 11) are radically different from each other (Fig. 3,
b and c). Furthermore, neither the TMDs of the
MsbA dimer nor the TMDs of BtuCD can be modeled onto the nf-P-gp TMD
structure determined here (note, the MsbA and BtuCD structures were
obtained in the absence of bound nucleotide). Confidence in the P-gp
structure, although at lower resolution, comes from the fact that a
similar structure was obtained by the very different methods of single
particle imaging and electron crystallography and that the protein used
was shown to be almost fully active both in drug binding and ATP
hydrolysis (27-29). The TMDs of two separated MsbA monomers could,
however, readily be mapped onto the TMDs of nf-P-gp (Fig. 4) if they
were rotated away from the dimer interface suggested by the original
MsbA crystal structure (11). Other considerations suggest that the
crystallographic dimer interface reported for MsbA may not reflect the
in vivo dimer interface (38, 39). The question of whether
the packing of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices per TMD, but there are notable exceptions with additional predicted transmembrane
-helices (9, 10). The
structures of two complete bacterial ABC transporters (9, 11) have
confirmed that the membrane-spanning segments are indeed
-helical,
although the packing of these
-helices within the membrane differs
markedly between the two structures.
-helices, and opening a
central pore along its length, potentially facilitating movement of
hydrophobic compounds from the lipid bilayer to the aqueous pore
of the transporter.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) is ~120o;
therefore, in principle there are 12 alternative orientations in which
each crystal can be merged with the core data set, of which only one
will be correct. A procedure was established in which structure factors
for the three alternative lattice refinement options were generated.
These were each tested against the core data set with rotation and/or
flipping options. The correct orientation was determined on the basis
of (a) inter-image phase residual (with the correct
orientation having a significantly lower residual than the nearest
alternative) and (b) examination of the projection map of
the untilted version of the crystal (e.g. identification of
the strong but narrow density in the P-gp-AMP-PNP structure, as
discussed under "Results"). In ~95% of the cases there was a
single orientation that was better than all the others in terms of
interimage phase residual. None of the crystals tested merged best with
a `flipped' orientation, implying that the crystals preferentially
adhere by only one face to the support film (see the "Results" and
"Discussion" sections). The three-dimensional maps were generated
using the CCP4 software (34), and modeling was carried out using XFIT
within the XTALVIEW software suite (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices remained unresolved.
Crystallographic image processing data for P-gp-AMP-PNP and nf-P-gp (in
parentheses)
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Fig. 1.
Quality of the three-dimensional data.
Plots of observed phases along selected lattice lines for the
P-gp-AMP-PNP crystals (filled circles) and the interpolated
lines used for extracting structure factors. Scatter is greater for the
weaker reflection (e.g. h,k = 1,2) at higher
resolution. Mean relative amplitudes for (1,2),
(2,-1), and (1,-2) reflections were 76, 1368, and
1035, respectively (arbitrary units).
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Fig. 2.
Comparison of nf-P-gp and P-gp-AMP-PNP
three-dimensional structures. A, stereo pair of
the nf-P-gp three-dimensional structure, displayed using netting at 1.0 (red) and 1.5
(yellow) above the mean
density level and viewed perpendicular to the crystal plane from the
more heavily stained side (corresponding to the extracellular surface).
B, equivalent views of the P-gp-AMP-PNP structure. The
arrow indicates the gap along one side of the central pore.
The locations of the three discrete densities A,
B, and C are indicated. C, stereo pair
of a side view of P-gp-AMP-PNP with the same color scheme as above. The
directions of the principle crystallographic axes a and
b are shown. Scale bar = 2.2 nm.
-helix (see below).
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Fig. 3.
Comparison of ABC transporters. The
three-dimensional structures of nf-P-gp (a) and P-gp-AMP-PNP
(d) are compared with the published structures of MsbA
(b) and BtuCD (c), all to the same scale. For
each protein a side view (top panels) and a top view
(looking down onto the equivalent of the extracellular surface)
(bottom panels) are shown. Yellow mesh shows
protein at ~1.0 above mean density level in (a) and
(d). a, Nf-P-gp is viewed along the main barrel
of protein density after rotation of the molecule to correct for the
slight tilting in the crystal (see "Results"). To achieve
this, the structure shown in Fig. 2 was rotated by 26 o
with respect to the horizontal axis and by 8 o about the
vertical axis. b, MsbA structure with six transmembrane
-helices per TMD. White and yellow C
traces
show separate MsbA monomers, connecting at the top via the TMDs and
with widely separated NBDs at the bottom. c, BtuCD
(blue C
traces) with two TMDs each containing 10
-helices. The NBDs and TMDs are closely associated. d,
the P-gp-AMP-PNP structure. The P-gp structures are distinct from both
MsbA and BtuCD in the putative transmembrane regions. Note that the
MsbA and BtuCD structures were in the nucleotide-free form.
-helices of BtuCD could not be readily
modeled onto the nf-P-gp map. This is presumably, in part, because the
BtuCD TMDs contain a total of 20 transmembrane
-helices, in contrast
to the 12 of P-gp. However, it should be noted that the lack of
sequence similarity is such that it is not possible to determine which
-helices, if any, of P-gp correspond to which of BtuCD. Similarly,
the TMDs of the intact MsbA homodimer (MsbA is equivalent to a
half-molecule of P-gp, and a homodimer of two monomers is believed to
form the functional molecule) could not be fitted to the P-gp densities (Fig. 3). However, making the assumption that the dimer interface in
the MsbA crystals is not the natural interface (38, 39; see also under
"Discussion"), the transmembrane regions of two separated MsbA
monomers could readily be modeled into the high density (TMD) region of
nf-P-gp (Fig. 4). The `arcuate'
arrangement of the
-helices in each of the two MsbA monomers almost
exactly forms the barrel shape of the TMDs of nf-P-gp. The nf-P-gp map
is slightly larger than the volume occupied by two MsbA monomers,
probably because of different resolution thresholds for the electron
versus x-ray crystallography data.
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Fig. 4.
Modeling of nf-P-gp with 2 × 6 transmembrane -helices. The
nf-Pgp structure (yellow netting) accommodates 2 × 6 transmembrane
-helices (red C
traces) in a
pseudo-symmetrical arrangement with a good fit. Two monomers of MsbA
were used to provide the 2 × 6 transmembrane
-helices after
removal of the NBDs. The nf-P-gp structure is displayed as in Fig. 3,
while the truncated MsbA monomers have been separately rotated and
translated for the fitting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices within the membrane. They show the transformation of a cylindrical, barrel-like structure into three discrete domains, one of which is
slightly smaller than the other two but longer perpendicular to the
membrane. This conformational change opens one side of the pore
throughout much of its length, equivalent to most of the depth of
the lipid bilayer. In a membrane environment, this would create access
from the lipid bilayer to the central pore. Because hydrophobic drugs
interact with P-gp from the lipid phase (40-42), this suggests a model
in which the TMDs part to enable hydrophobic drugs in the bilayer to
enter the central pore prior to extrusion, rather than a model in which
the drug moves across the membrane along a lipid-protein interface at
the outer surface of the P-gp molecule. The conformational changes
observed are consistent with a `helix rotation' model for transport
(39) but would be difficult to square with a `tilting helix' model (11). Finally, it is significant that major conformational change occurs upon ATP binding rather than ATP hydrolysis. Although it has
often been assumed that ATP hydrolysis drives the transport process,
recent biochemical data show that reductions in drug binding affinity
to P-gp are also due to ATP binding rather than hydrolysis (17, 18,
28). Thus, ATP binding appears to drive the major conformational
changes that reduce drug binding affinity and expose the drug binding
site to the extracellular milieu (central aqueous pore); ATP hydrolysis
may therefore simply `reset' the transporter (14).
-helices in the monomer of MsbA actually reflects
that of P-gp awaits more sophisticated
modeling and a higher resolution structure for P-gp. Cross-linking data
(43)2 already suggest that there will be important
differences. Nevertheless, the present data do show that the densities
of the TMDs of nf-P-gp are entirely consistent with a pseudosymmetric
structure of 2 × 6 transmembrane
-helices arranged to form a
barrel (Fig. 4).
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ACKNOWLEDGEMENTS |
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We thank Drs. K. J. Linton (Medical Research Council Clinical Sciences Centre, London) and I. Kerr (University of Nottingham) for helpful discussions, Drs. J. P. Derrick and S. Prince (Biomolecular Sciences, University of Manchester Institute of Science and Technology) for help with the XtalView software, and Dr. K. Sidhu and Dr. S. J. Hubbard and S. Oliver (Biomolecular Sciences, UMIST) for computer support.
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FOOTNOTES |
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* This work was supported by Cancer Research UK.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.
§ Both authors contributed equally to the work.
¶ Recipient of a scholarship from the Borno State Government of Nigeria.
To whom correspondence should be addressed. Tel.: 44-161-200 4187; Fax: 44-161-236-0409; E-mail: r.ford@umist.ac.uk.
Published, JBC Papers in Press, December 25, 2002, DOI 10.1074/jbc.M211758200
2 D. R. Stenham, J. D. Campbell, M. S. P. Sansom, C. F. Higgins, I. D. Kerr, and K. J. Linton, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ABC, ATP binding cassette; TMD, transmembrane domain; NBD, nucleotide binding domain; P-gp, P-glycoprotein; nf-P-gp, nucleotide-free P-gp.
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