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Structure of the multidrug resistance P-glycoprotein
seminars in C A N C E R B I OLOG Y, Vol 8, 1997: pp 135]142
Structure of the multidrug resistance P-glycoprotein Christopher F. HigginsU , Richard CallaghanU , Kenneth J. LintonU , Mark F. Rosenberg† and Robert C. Ford† regulate the activity of heterologous ion channels,1,2 although it is not yet known how this regulation is achieved. In order to understand the molecular mechanisms of P-gp-mediated drug transport, and in particular the determinants of substrate specificity, structural data are required. Direct structural analysis of membrane proteins is difficult and much of our understanding of the structure of P-gp comes from indirect experimental approaches and by analogy with other ABC transporters. Recently, however, a low resolution structure for P-gp has been reported.3 In this article we review these biochemical and genetic data, placing them in the context of these new structural insights.
In order to elucidate the mechanism by which the multidrug resistance P-glycoprotein extrudes cytotoxic drugs from the cell, and particularly the number and nature of the drug binding site(s), knowledge of the structure of P-gp is essential. A considerable body of genetic and biochemical data has accrued which gives insights into P-gp structure and function. These data are critically reviewed, particularly in relation to the low resolution structure of P-gp which has recently been determined by electron microscopy. P-gp is one of the best characterised of the ABC transporters and these structure-function studies may have more general implications. Key words: P-glycoprotein r protein structure r membrane protein r ABC transporters Q1997 Academic Press Ltd
Domain organisation Introduction
When the primary sequence of P-gp was determined, over 10 years ago,4 ] 6 analogy with well-characterised bacterial ABC transporters led to the prediction that P-gp consists of four distinct domains: two highly hydrophobic integral membrane domains and two hydrophilic nucleotide-binding domains ŽNBDs. located at the cytoplasmic face of the membrane ŽFigure 1.. Subsequent genetic and biochemical data are consistent with this organisation. P-gp can be viewed as two half molecules, each consisting of one integral membrane domain and one nucleotide-binding domain; the amino acid sequences of the two half molecules are closely related to each other suggesting a pseudosymmetry to the protein structure. The two half molecules are separated by a highly charged ‘linker’ region which is phosphorylated at several sites by protein kinase C in vivo and in vitro.7 Phosphorylation of this ‘linker’ does not appear to be required for active transport 8,9 but modulates the
THE MAMMALIAN MULTIDRUG resistance P-glycoprotein ŽP-gp . is probably the best characterised ABC transporter. Its primary activity is to mediate the transmembrane translocation of hydrophobic molecules, including many cytotoxic drugs, although whether its role is simply to protect against exogenous toxins, or to transport an unknown endogenous substrateŽs., is still the subject of debate. In addition to being an active transporter, P-gp can also U
From the Nuffield Department of Clinical Biochemistry, and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK and †Department of Biochemistry and Applied Molecular Biology, UMIST, P.O. Box 88, Manchester M60 1QD UK Q1997 Academic Press Ltd 1044-579Xr 97r 030135q 08$25.00r 0r se970067
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Figure 1. Topological map and domain organisation of P-gp, predicted from its primary sequence.
ability of P- gp to regulate heterologous ion channels.10
How, then, can one explain several studies which report ‘alternative’ topologies for the integral membrane domains of P-gp? For example, C-terminally truncated domains expressed in vitro and inserted into microsomal membranes can adopt both the standard six-plus-six topology and an ‘alternative’ topology in which two of the predicted transmembrane segments fail to insert into the membrane.16,17 Similarly, expression of C-terminally truncated P-gp molecules in Xenopus oocytes generates ‘alternative’ transmembrane topologies.18,19 Finally, expression of C-terminally truncated P-gp in E. coli, fused to either b-lactamase or alkaline phosphatase, generates data inconsistent with the ‘six-plus-six’ model and indicates two distinct topologies different from those reported elsewhere.20 ] 22 Of course, it is possible that P-gp can exist in more than one configuration, each with a distinct transmembrane topology. Indeed, it has been suggested that interconversion between these topologies may be related to the mechanism of action of P-gp.17 However, for several reasons it is perhaps more likely that P-gp adopts a single transmembrane topology and that the alternative topologies observed are a consequence of experimental perturbation of the protein. First, the studies describing ‘alternative’ topologies do not generate a single consistent model. Second, the transmembrane topology of P-gp is easily perturbed: single amino acid changes can alter the topology;17,22 and each study includes examples where inclusion of specific P-gp sequence influenced the preceding transmembrane topology, implying a role for C-terminal sequences in establishing or maintaining the transmembrane topology. Consistent with this is the finding that insertion of transmembrane segments requires that they interact cooperatively.18,19 Third,
Integral membrane domains The integral membrane domains of P-gp have two central roles in the transport process. First, they form the pathway through which solute is translocated across the membrane Žalthough parts of the nucleotide-binding domain could also, potentially, contribute to this pathway.. Second, they provide the amino acid residues which interact directly with the substrate and form the substrate binding-siteŽs.: the integral membrane domains can be photoaffinitylabelled by drug analogues, and mutations altering substrate specificity map to these domains Žsee article by Ueda et al, this volume.. Based on primary sequence data, most standard algorithms predict that each integral membrane domain of P-gp consists of six membrane-spanning ahelices separated by hydrophilic loops, a total of 12 membrane-spanning a-helices per molecule Žthe ‘six-plus-six’ model; Figure 1.. Two recent studies provide strong support for this topology. In these studies the intra- or extracellular location of short epitopes11 or cysteine residue,12 introduced at defined points in the P-gp sequence, was determined. Importantly, minimal perturbation of the protein sequence ensured that the modified proteins were active and could be assumed to be in the native conformation. Studies using antibodies directed against defined extracellular epitopes of P-gp are also consistent with the six-plus-six model.13 ] 15 Finally, recent low resolution structural data3 can best be interpreted in terms of the ‘six-plus-six’ model Žsee below.. 136
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all of the studies suggesting an alternative topology involved C-terminally-truncated proteins expressed in heterologous membrane systems Žmicrosomes, oocytes, E. coli .. In none of these studies was it possible to determine whether P-gp was active. In contrast, the studies demonstrating a ‘six-plus-six’ topology were carried out on full length, active P-gp. Although it now seems reasonable to accept that the ‘six-plus-six’ model is essentially correct, the possibility that conformational changes during the transport cycle may include gross changes in transmembrane topology cannot be excluded. However, even accepting the ‘six-plus-six’ topology, the precise limits of each membrane-spanning segment Ži.e., which amino acids are embedded within the bilayer and which are exposed to the aqueous phase. are not known. Furthermore, it has not been formally demonstrated that the membrane-spanning segments are actually a-helical, although analogy with other membrane proteins and modelling studies strongly suggests that they are ŽKerr et al, unpublished results.. Consistent with this, Fourier transform infrared spectroscopy shows that P-gp is 32% a-helical. 23 The integral membrane domains of P-gp form the binding siteŽs. for transported substrates and allosteric regulators, yet the residues which directly interact with substrate remain to be defined. Many mutations which alter substrate specificity have been isolated Žsee Ueda et al, this volume. but, as the amino acid changes are scattered throughout the membrane-spanning a-helices and the loops which separate them, it has not been possible to define a substrate-binding pocket and the data may best be interpreted in terms of general structural perturbations. Cross-linking studies have also failed to define residues directly involved in substrate interactions, although they do demonstrate that residues from both halves of the P-gp molecule are involved.24,25 The precise number of substrate-binding sites has also been an issue of some contention, and many published studies are hard to interpret. The most rigorous pharmacological analysis of drug binding to P-gp suggests the presence of at least two drug interaction sites, a site for transported drugs such as vinblastine, and a second allosterically-coupled site at which certain transport modulators interact.26 ] 28 Much remains to be learnt about the nature of these sites and their precise location on the P-gp molecule. In order to explain the broad specificity of P-gp, imaginative models involving a large number of binding sites, or hydrophobic ‘surfaces’ with which drugs interact, have been proposed. However, there is no
need to invoke such models. Several examples of multispecific substrate binding Žeg. the bacterial oligopeptide transporter,29,30 and the mammalian odorant binding protein31 . have been shown, by high-resolution structural studies, to involve defined but promiscuous binding sites. Furthermore, evidence that substrates gain access to P-gp from the lipid phase can explain the exclusion of many nonhydrophobic cellular constituents from such a promiscuous site.32
Nucleotide-binding domains The two nucleotide-binding domains ŽNBDs. of P-gp share 30]40% amino acid sequence identity with each other and the equivalent domains of other ABC transporters. These domains bind and hydrolyse ATP and couple the hydrolysis of ATP to solute translocation across the membrane Žsee Senior and Gadsby, this volume.. Although the mechanism of energy transduction is unknown, it is likely to involve ATP hydrolysis- induced conformational changes: the binding of substrates to P-gp is known to stimulate ATP hydrolysis33,34 and conformational changes occur following drug binding and ATP hydrolysis.23,35 At the primary sequence level, the NBDs include the so-called Walker motifs, characteristic of many nucleotide-binding proteins. They also share significant additional sequence identity, in particular a short ‘signature’ motif which defines the NBDs of ABC transporters.36 Despite the fact that they are relatively hydrophilic, the isolated NBDs have proved difficult to express and purify in a fully active form and no structural data for any NBD have yet been obtained. The NBDs have, however, been modelled based on the known structures of nucleotide-binding proteins such as adenylate kinase and ras.36,37 These two models are rather similar and are both consistent with mutational analysis of the NBDs of ABC transporters. In particular, mutagenesis and ATP or nucleotide analogue binding studies show that, as predicted, the Walker motifs function in ATP binding and hydrolysis. The NBDs are located at the cytoplasmic face of the membrane, consistent with a role in binding and hydrolysing ATP. However, for several bacterial ABC transporters the NBDs have been shown to be accessible to biochemical reagents from the extracellular surface of the membrane.38 ] 40 As there is no reason to suppose that the overall architecture of P-gp differs significantly from that of the bacterial ABC 137
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transporters, the NBDs of P-gp may also be accessible from the extracellular face of the membrane. This accessibility has been interpreted to indicate that a loop of the NBD spans the bilayer directly, or via a ‘pore’ formed by the transmembrane domains.38 However, as the transmembrane domains of P-gp appear to form a large, 5-nm diameter aqueous pore spanning much of the membrane Žsee below., an alternative and perhaps, more likely model is that the NBDs are entirely intracellular but are accessible from the extracellular face of the membrane through this pore. The resolution of this question is an important issue because it reflects on whether or not the NBDs may contribute to the transmembrane pore.
get size of 250 kDa, corresponding most closely to a dimer of two 140-kDa protein molecules Žthis method does not detect carbohydrate..42 However, this method relies on protein target size which may not reflect molecular mass for an irregularly shaped protein: it is now known that P-gp is loosely packed Žsee below.. P-gp dimers or oligomers have also been implicated by chemical cross-linking and ultracentrifugation studies.43,44 However, none of these studies can distinguish between dimeric P-gp and a complex between monomeric P-gp and another protein, and they cannot ascertain whether a dimer is the minimum functional unit. The suggestion that dimerisation is influenced by the phosphorylation state of P-gp 44 is intriguing, but cannot be interpreted in terms of the regulation of drug transport, which appears to be phosphorylation independent.8,9 Perhaps this reflects interactions with heterologous proteins as phosphorylation is known to influence the ability of P-gp to regulate the activity of heterologous ion channels.10
Oligomeric state of P-gp The oligomeric state of P-gp has been the subject of some debate, a consequence of the imperfect methods available and the particular problems associated with studying a large membrane protein. For example, in vivo studies of membranes in which P-gp is substantially overproduced may reflect inappropriate protein aggregation. In vitro studies suffer from potential artefacts of detergent solubilisation. Furthermore, many studies have not sought to distinguish between the minimum functional unit, and the number of these functional units which might associate into a larger complex. Studies on the oligomeric state of other ABC proteins have given few clues. The balance of recent data now point towards a monomer as the minimum functional unit, although these data do not exclude the possibility that these monomers might form dimers or larger oligomers in some circumstances, or even that oligomerisation might alter the properties of each individual functional monomer. Thus, careful biochemical crosslinking, and immunoprecipitation studies on purified P-gp indicate that the minimum functional unit is a monomer 41 ŽTaylor and Higgins, unpublished.. Electron microscopic observation of purified, active P-gp labelled with lectin conjugated to a gold particle showed only a single gold particle per protein, indicating that the protein is monomeric,3 although labelling of a dimer could be subject to steric interference. Finally, the low resolution structure of P-gp Žsee below. is consistent with a monomer.3 In all these studies P-gp was active in drug binding and ATP hydrolysis. In apparent contrast, radiation inactivation using P-gp-overexpressing cell membranes indicates a tar-
A low resolution structure for P-gp
˚ resolution structure for P-gp was Recently, a 25-A obtained by electron microscopy and single-particle image analysis of both detergent solubilised and lipid reconstituted P-gp. 3 The structure was further refined by three-dimensional reconstructions from single particle images and by Fourier projection maps of small two-dimensional crystalline arrays. A diagrammatic representation of the structure is shown in Figure 2. This structure can be interpreted in terms of the biochemical and genetic data discussed above and is consistent with many preconceptions of what the protein ‘should look like’. However, it must be remembered that no direct correlation between any structural feature observed and primary sequence has yet been obtained. When viewed from the extracellular face of the membrane the protein is toroidal with a protein ring of diameter about 10 nm surrounding a large central pore ŽFigure 3.. The protein ring has two important features. First, it exhibits sixfold symmetry: a model in which the six lobes correspond to the six extracellular loops between pairs of a-helices is compelling but has yet to be confirmed experimentally. Second, a ‘gap’ may be present in the protein ring, potentially providing access between the central pore and the lipid phase: the significance of this is discussed below. The central pore appears to be aqueous, as it stains 138
Structure of the multidrug resistance P-glycoprotein
a-helices. This suggests that the ‘business end’ of the molecule is at the cytoplasmic face of the membrane, consistent with data showing that the substrate binding site of P-gp is accessible from the inner face of the membrane,47 that substrates can be cross-linked to regions of the protein predicted to be at the cytoplasmic face of the membrane,48 and that several mutations altering amino acids at the cytoplasmic face of the membrane can influence substrate selectivity 49,50 As discussed above, mutations which alter substrate specificity mapping to other parts of the protein may have an indirect effect through general changes in protein structure. Although the paths of the transmembrane a-helices cannot be traced at this low resolution, it is interesting to consider how they may be organised to generate a chamber within the membrane. Modelling suggests that, with appropriate tilting, the 12 membrane spanning a-helices of P-gp can generate such a structure ŽKerr and Higgins, unpublished., and hexameric annexin is of similar molecular mass and has approximately the same dimensions with a large central pore.51 The two related halves of P-gp could be organised in true twofold symmetry Žwith helix 1 neighbouring helix 12 and helix 7 neighbouring helix 6., or in mirror symmetry Žwith helices 1 and 7 and helices 6 and 12 as neighbours. ŽFigure 4.. Cross-linking studies showing a proximity between helix 6 and helix 12 suggest the latter organisation.35 The two intracellular lobes of P-gp which presumably correspond to the NBDs are asymetrically organised in the structure and do not appear to interact with each other. Consistent with this, no interaction between the NBDs can be detected genetically using the yeast two-hybrid system ŽBegley et al, in preparation.. However, the catalytic cycles of the two NBDs are coupled Žsee Senior and Gadsby, this volume., and this has been taken to indicate that the
Figure 2. Cartoon of the three-dimensional structure of ˚ resolution by electron microsP-gp determined to 25 A copy.3 Cartoon courtesy of Kevin Hannavy.
Figure 3. Projection map of P-gp viewed from above the extracellular face of the membrane, showing a protein ring surrounding a large aqueous pore. Note the sixfold symmetry and the putative ‘gap’ in the protein ring providing access between the central pore and the lipid phase.
intensely with uranyl acetate and phosphotungstate. The pore is large, about 5 nm in diameter, and is the entrance to a cup-shaped chamber within the membrane, presumably formed by the twelve membranespanning a-helices ŽFigure 2.. An entry to this chamber from the lipid phase of the membrane is apparent, consistent with evidence that P-gp acts as a ‘flippase’ with drug substrates gaining access to their binding siteŽs. from the inner leaflet of the lipid bilayer.32,45,46 This chamber narrows as it passes through the membrane and is closed at the cytoplasmic face of the membrane, presumably by sequences from the nucleotide-binding domains and the cytoplasmic loops separating the transmembrane
Figure 4. Two possible orientations of the transmembrane helices forming the transmembrane pore of P-gp: the twofold symmetry model ŽA. or the mirror symmetry model ŽB..
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two domains interact directly. It is possible that direct interactions between the NBDs only occur in the presence of ATP andror substrate, and were therefore not apparent in the structure determined in the absence of these ligands. Alternatively, coupling between the NBDs may be mediated indirectly through interactions with the transmembrane domains. This is an important issue to resolve as it directly pertains to the mechanism of energy transduction. The structure of P-gp is very different from the low resolution structures determined for two other transporting ATPases, the Ca2q-ATPase and the HqATPase. In these two proteins the transmembrane a-helices are relatively tightly packed, rather than forming a large transmembrane pore. However, like P-gp, the complex which mediates protein translocation across the endoplasmic reticulum also has a 4]6-nm pore.52 The structure determined for P-gp is consistent with a general architecture for ABC transporters. The substrates transported by different ABC transporters can vary widely, from small ions to large polypeptides and polysaccharides:53 a common architecture with a large pore could readily be adapted to accommodate different sized substrates with relatively minor changes to the ‘gate’ at the cytoplasmic face of the membrane. Additionally, one ABC transporter ŽCFTR. is an ion channel and, again, the structure of P-gp is compatible with adaptation to such a function: indeed, studies on CFTR have led to the suggestion that several helices contribute to an aqueous pore gated at the cytoplasmic face of the membrane consistent with the structure determined for P-gp 54 ŽAkabas, M., personal communication.. Finally, it should be remembered that the structural data obtained for P-gp reflect a static conformation, yet the structure of P-gp is likely to be dynamic. It is generally accepted that P-gp undergoes significant conformational changes during its transport cycle, and ATP hydrolysis by the NBDs must induce conformational changes in the transmembrane domains to achieve active transport. Changes in crosslinking between cysteine residues introduced into P-gp imply a conformational change upon drug binding,35 and Fourier transform infrared spectroscopy shows that MgATP induces conformational changes involving a significant portion of P-gp. 23 These conformational changes do not involve a gross perturbation of structure, as the proportions of a-helix and b-sheet remain constant, and therefore presumably involve a movement of secondary structural elements or domains with respect to each other. Nevertheless,
the dynamic aspects of protein structure are essential for any understanding of its mechanism of action.
Prospects Although the direct determination of a structure for P-gp is an important step in understanding the mechanisms of transport, the resolution is low and much remains to be learned. Electron diffraction from large two-dimensional crystals is likely to im˚ although prove the resolution, perhaps to 6]8 A, atomic level resolution seems unlikely to be achieved in the foreseeable future. Nevertheless, low level resolution structural analysis together with careful mutagenesis and biophysical studies should give considerable insight into how this protein works, with consequent implications for developing improved approaches to reversing multidrug resistance in the clinic.
Acknowledgements We are grateful to the Cancer Research Campaign, Imperial Cancer Research Fund, and the Medical Research Council for support. We thank Kevin Hannavy for preparing Figure 2. CFH is a Howard Hughes International Research Scholar.
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to colchicine in multidrug-resistant human cells conferred by gly-185-val-185 substitution in P-glycoprotein. Proc Natl Acad Sci ŽUSA. 87:7225]7229 Choi K, Chen C-J, Kriegler M, Roninson IB Ž1988. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the MDR1 ŽP-glycoprotein. gene. Cell 53:519]529. Luecke H, Chang BT, Mailliard WS, Schaepfer DD, Haigler HT Ž1995. Crystal-structure of the annexin-xii hexamer and implications for bilayer insertion. Nature 378:512]515 Hamman BD, Chen J-C, Johnson EE, Johnson AE Ž1997. The aqueous pore through the translocon has a diameter of 40]60 ˚ during cotranslational protein translocation at the ER memA brane. Cell 89:535]544 Higgins CF Ž1992. ABC transporters: from microorganisms to man. Ann Rev Cell Biol 8:67]113 Akabas MH, Kaufmann C, Cook TA, Archdeacon P Ž1994. Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 269:148]14868
Structure of the multidrug resistance P-glycoprotein Christopher F. HigginsU , Richard CallaghanU , Kenneth J. LintonU , Mark F. Rosenberg† and Robert C. Ford† regulate the activity of heterologous ion channels,1,2 although it is not yet known how this regulation is achieved. In order to understand the molecular mechanisms of P-gp-mediated drug transport, and in particular the determinants of substrate specificity, structural data are required. Direct structural analysis of membrane proteins is difficult and much of our understanding of the structure of P-gp comes from indirect experimental approaches and by analogy with other ABC transporters. Recently, however, a low resolution structure for P-gp has been reported.3 In this article we review these biochemical and genetic data, placing them in the context of these new structural insights.
In order to elucidate the mechanism by which the multidrug resistance P-glycoprotein extrudes cytotoxic drugs from the cell, and particularly the number and nature of the drug binding site(s), knowledge of the structure of P-gp is essential. A considerable body of genetic and biochemical data has accrued which gives insights into P-gp structure and function. These data are critically reviewed, particularly in relation to the low resolution structure of P-gp which has recently been determined by electron microscopy. P-gp is one of the best characterised of the ABC transporters and these structure-function studies may have more general implications. Key words: P-glycoprotein r protein structure r membrane protein r ABC transporters Q1997 Academic Press Ltd
Domain organisation Introduction
When the primary sequence of P-gp was determined, over 10 years ago,4 ] 6 analogy with well-characterised bacterial ABC transporters led to the prediction that P-gp consists of four distinct domains: two highly hydrophobic integral membrane domains and two hydrophilic nucleotide-binding domains ŽNBDs. located at the cytoplasmic face of the membrane ŽFigure 1.. Subsequent genetic and biochemical data are consistent with this organisation. P-gp can be viewed as two half molecules, each consisting of one integral membrane domain and one nucleotide-binding domain; the amino acid sequences of the two half molecules are closely related to each other suggesting a pseudosymmetry to the protein structure. The two half molecules are separated by a highly charged ‘linker’ region which is phosphorylated at several sites by protein kinase C in vivo and in vitro.7 Phosphorylation of this ‘linker’ does not appear to be required for active transport 8,9 but modulates the
THE MAMMALIAN MULTIDRUG resistance P-glycoprotein ŽP-gp . is probably the best characterised ABC transporter. Its primary activity is to mediate the transmembrane translocation of hydrophobic molecules, including many cytotoxic drugs, although whether its role is simply to protect against exogenous toxins, or to transport an unknown endogenous substrateŽs., is still the subject of debate. In addition to being an active transporter, P-gp can also U
From the Nuffield Department of Clinical Biochemistry, and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK and †Department of Biochemistry and Applied Molecular Biology, UMIST, P.O. Box 88, Manchester M60 1QD UK Q1997 Academic Press Ltd 1044-579Xr 97r 030135q 08$25.00r 0r se970067
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Figure 1. Topological map and domain organisation of P-gp, predicted from its primary sequence.
ability of P- gp to regulate heterologous ion channels.10
How, then, can one explain several studies which report ‘alternative’ topologies for the integral membrane domains of P-gp? For example, C-terminally truncated domains expressed in vitro and inserted into microsomal membranes can adopt both the standard six-plus-six topology and an ‘alternative’ topology in which two of the predicted transmembrane segments fail to insert into the membrane.16,17 Similarly, expression of C-terminally truncated P-gp molecules in Xenopus oocytes generates ‘alternative’ transmembrane topologies.18,19 Finally, expression of C-terminally truncated P-gp in E. coli, fused to either b-lactamase or alkaline phosphatase, generates data inconsistent with the ‘six-plus-six’ model and indicates two distinct topologies different from those reported elsewhere.20 ] 22 Of course, it is possible that P-gp can exist in more than one configuration, each with a distinct transmembrane topology. Indeed, it has been suggested that interconversion between these topologies may be related to the mechanism of action of P-gp.17 However, for several reasons it is perhaps more likely that P-gp adopts a single transmembrane topology and that the alternative topologies observed are a consequence of experimental perturbation of the protein. First, the studies describing ‘alternative’ topologies do not generate a single consistent model. Second, the transmembrane topology of P-gp is easily perturbed: single amino acid changes can alter the topology;17,22 and each study includes examples where inclusion of specific P-gp sequence influenced the preceding transmembrane topology, implying a role for C-terminal sequences in establishing or maintaining the transmembrane topology. Consistent with this is the finding that insertion of transmembrane segments requires that they interact cooperatively.18,19 Third,
Integral membrane domains The integral membrane domains of P-gp have two central roles in the transport process. First, they form the pathway through which solute is translocated across the membrane Žalthough parts of the nucleotide-binding domain could also, potentially, contribute to this pathway.. Second, they provide the amino acid residues which interact directly with the substrate and form the substrate binding-siteŽs.: the integral membrane domains can be photoaffinitylabelled by drug analogues, and mutations altering substrate specificity map to these domains Žsee article by Ueda et al, this volume.. Based on primary sequence data, most standard algorithms predict that each integral membrane domain of P-gp consists of six membrane-spanning ahelices separated by hydrophilic loops, a total of 12 membrane-spanning a-helices per molecule Žthe ‘six-plus-six’ model; Figure 1.. Two recent studies provide strong support for this topology. In these studies the intra- or extracellular location of short epitopes11 or cysteine residue,12 introduced at defined points in the P-gp sequence, was determined. Importantly, minimal perturbation of the protein sequence ensured that the modified proteins were active and could be assumed to be in the native conformation. Studies using antibodies directed against defined extracellular epitopes of P-gp are also consistent with the six-plus-six model.13 ] 15 Finally, recent low resolution structural data3 can best be interpreted in terms of the ‘six-plus-six’ model Žsee below.. 136
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all of the studies suggesting an alternative topology involved C-terminally-truncated proteins expressed in heterologous membrane systems Žmicrosomes, oocytes, E. coli .. In none of these studies was it possible to determine whether P-gp was active. In contrast, the studies demonstrating a ‘six-plus-six’ topology were carried out on full length, active P-gp. Although it now seems reasonable to accept that the ‘six-plus-six’ model is essentially correct, the possibility that conformational changes during the transport cycle may include gross changes in transmembrane topology cannot be excluded. However, even accepting the ‘six-plus-six’ topology, the precise limits of each membrane-spanning segment Ži.e., which amino acids are embedded within the bilayer and which are exposed to the aqueous phase. are not known. Furthermore, it has not been formally demonstrated that the membrane-spanning segments are actually a-helical, although analogy with other membrane proteins and modelling studies strongly suggests that they are ŽKerr et al, unpublished results.. Consistent with this, Fourier transform infrared spectroscopy shows that P-gp is 32% a-helical. 23 The integral membrane domains of P-gp form the binding siteŽs. for transported substrates and allosteric regulators, yet the residues which directly interact with substrate remain to be defined. Many mutations which alter substrate specificity have been isolated Žsee Ueda et al, this volume. but, as the amino acid changes are scattered throughout the membrane-spanning a-helices and the loops which separate them, it has not been possible to define a substrate-binding pocket and the data may best be interpreted in terms of general structural perturbations. Cross-linking studies have also failed to define residues directly involved in substrate interactions, although they do demonstrate that residues from both halves of the P-gp molecule are involved.24,25 The precise number of substrate-binding sites has also been an issue of some contention, and many published studies are hard to interpret. The most rigorous pharmacological analysis of drug binding to P-gp suggests the presence of at least two drug interaction sites, a site for transported drugs such as vinblastine, and a second allosterically-coupled site at which certain transport modulators interact.26 ] 28 Much remains to be learnt about the nature of these sites and their precise location on the P-gp molecule. In order to explain the broad specificity of P-gp, imaginative models involving a large number of binding sites, or hydrophobic ‘surfaces’ with which drugs interact, have been proposed. However, there is no
need to invoke such models. Several examples of multispecific substrate binding Žeg. the bacterial oligopeptide transporter,29,30 and the mammalian odorant binding protein31 . have been shown, by high-resolution structural studies, to involve defined but promiscuous binding sites. Furthermore, evidence that substrates gain access to P-gp from the lipid phase can explain the exclusion of many nonhydrophobic cellular constituents from such a promiscuous site.32
Nucleotide-binding domains The two nucleotide-binding domains ŽNBDs. of P-gp share 30]40% amino acid sequence identity with each other and the equivalent domains of other ABC transporters. These domains bind and hydrolyse ATP and couple the hydrolysis of ATP to solute translocation across the membrane Žsee Senior and Gadsby, this volume.. Although the mechanism of energy transduction is unknown, it is likely to involve ATP hydrolysis- induced conformational changes: the binding of substrates to P-gp is known to stimulate ATP hydrolysis33,34 and conformational changes occur following drug binding and ATP hydrolysis.23,35 At the primary sequence level, the NBDs include the so-called Walker motifs, characteristic of many nucleotide-binding proteins. They also share significant additional sequence identity, in particular a short ‘signature’ motif which defines the NBDs of ABC transporters.36 Despite the fact that they are relatively hydrophilic, the isolated NBDs have proved difficult to express and purify in a fully active form and no structural data for any NBD have yet been obtained. The NBDs have, however, been modelled based on the known structures of nucleotide-binding proteins such as adenylate kinase and ras.36,37 These two models are rather similar and are both consistent with mutational analysis of the NBDs of ABC transporters. In particular, mutagenesis and ATP or nucleotide analogue binding studies show that, as predicted, the Walker motifs function in ATP binding and hydrolysis. The NBDs are located at the cytoplasmic face of the membrane, consistent with a role in binding and hydrolysing ATP. However, for several bacterial ABC transporters the NBDs have been shown to be accessible to biochemical reagents from the extracellular surface of the membrane.38 ] 40 As there is no reason to suppose that the overall architecture of P-gp differs significantly from that of the bacterial ABC 137
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transporters, the NBDs of P-gp may also be accessible from the extracellular face of the membrane. This accessibility has been interpreted to indicate that a loop of the NBD spans the bilayer directly, or via a ‘pore’ formed by the transmembrane domains.38 However, as the transmembrane domains of P-gp appear to form a large, 5-nm diameter aqueous pore spanning much of the membrane Žsee below., an alternative and perhaps, more likely model is that the NBDs are entirely intracellular but are accessible from the extracellular face of the membrane through this pore. The resolution of this question is an important issue because it reflects on whether or not the NBDs may contribute to the transmembrane pore.
get size of 250 kDa, corresponding most closely to a dimer of two 140-kDa protein molecules Žthis method does not detect carbohydrate..42 However, this method relies on protein target size which may not reflect molecular mass for an irregularly shaped protein: it is now known that P-gp is loosely packed Žsee below.. P-gp dimers or oligomers have also been implicated by chemical cross-linking and ultracentrifugation studies.43,44 However, none of these studies can distinguish between dimeric P-gp and a complex between monomeric P-gp and another protein, and they cannot ascertain whether a dimer is the minimum functional unit. The suggestion that dimerisation is influenced by the phosphorylation state of P-gp 44 is intriguing, but cannot be interpreted in terms of the regulation of drug transport, which appears to be phosphorylation independent.8,9 Perhaps this reflects interactions with heterologous proteins as phosphorylation is known to influence the ability of P-gp to regulate the activity of heterologous ion channels.10
Oligomeric state of P-gp The oligomeric state of P-gp has been the subject of some debate, a consequence of the imperfect methods available and the particular problems associated with studying a large membrane protein. For example, in vivo studies of membranes in which P-gp is substantially overproduced may reflect inappropriate protein aggregation. In vitro studies suffer from potential artefacts of detergent solubilisation. Furthermore, many studies have not sought to distinguish between the minimum functional unit, and the number of these functional units which might associate into a larger complex. Studies on the oligomeric state of other ABC proteins have given few clues. The balance of recent data now point towards a monomer as the minimum functional unit, although these data do not exclude the possibility that these monomers might form dimers or larger oligomers in some circumstances, or even that oligomerisation might alter the properties of each individual functional monomer. Thus, careful biochemical crosslinking, and immunoprecipitation studies on purified P-gp indicate that the minimum functional unit is a monomer 41 ŽTaylor and Higgins, unpublished.. Electron microscopic observation of purified, active P-gp labelled with lectin conjugated to a gold particle showed only a single gold particle per protein, indicating that the protein is monomeric,3 although labelling of a dimer could be subject to steric interference. Finally, the low resolution structure of P-gp Žsee below. is consistent with a monomer.3 In all these studies P-gp was active in drug binding and ATP hydrolysis. In apparent contrast, radiation inactivation using P-gp-overexpressing cell membranes indicates a tar-
A low resolution structure for P-gp
˚ resolution structure for P-gp was Recently, a 25-A obtained by electron microscopy and single-particle image analysis of both detergent solubilised and lipid reconstituted P-gp. 3 The structure was further refined by three-dimensional reconstructions from single particle images and by Fourier projection maps of small two-dimensional crystalline arrays. A diagrammatic representation of the structure is shown in Figure 2. This structure can be interpreted in terms of the biochemical and genetic data discussed above and is consistent with many preconceptions of what the protein ‘should look like’. However, it must be remembered that no direct correlation between any structural feature observed and primary sequence has yet been obtained. When viewed from the extracellular face of the membrane the protein is toroidal with a protein ring of diameter about 10 nm surrounding a large central pore ŽFigure 3.. The protein ring has two important features. First, it exhibits sixfold symmetry: a model in which the six lobes correspond to the six extracellular loops between pairs of a-helices is compelling but has yet to be confirmed experimentally. Second, a ‘gap’ may be present in the protein ring, potentially providing access between the central pore and the lipid phase: the significance of this is discussed below. The central pore appears to be aqueous, as it stains 138
Structure of the multidrug resistance P-glycoprotein
a-helices. This suggests that the ‘business end’ of the molecule is at the cytoplasmic face of the membrane, consistent with data showing that the substrate binding site of P-gp is accessible from the inner face of the membrane,47 that substrates can be cross-linked to regions of the protein predicted to be at the cytoplasmic face of the membrane,48 and that several mutations altering amino acids at the cytoplasmic face of the membrane can influence substrate selectivity 49,50 As discussed above, mutations which alter substrate specificity mapping to other parts of the protein may have an indirect effect through general changes in protein structure. Although the paths of the transmembrane a-helices cannot be traced at this low resolution, it is interesting to consider how they may be organised to generate a chamber within the membrane. Modelling suggests that, with appropriate tilting, the 12 membrane spanning a-helices of P-gp can generate such a structure ŽKerr and Higgins, unpublished., and hexameric annexin is of similar molecular mass and has approximately the same dimensions with a large central pore.51 The two related halves of P-gp could be organised in true twofold symmetry Žwith helix 1 neighbouring helix 12 and helix 7 neighbouring helix 6., or in mirror symmetry Žwith helices 1 and 7 and helices 6 and 12 as neighbours. ŽFigure 4.. Cross-linking studies showing a proximity between helix 6 and helix 12 suggest the latter organisation.35 The two intracellular lobes of P-gp which presumably correspond to the NBDs are asymetrically organised in the structure and do not appear to interact with each other. Consistent with this, no interaction between the NBDs can be detected genetically using the yeast two-hybrid system ŽBegley et al, in preparation.. However, the catalytic cycles of the two NBDs are coupled Žsee Senior and Gadsby, this volume., and this has been taken to indicate that the
Figure 2. Cartoon of the three-dimensional structure of ˚ resolution by electron microsP-gp determined to 25 A copy.3 Cartoon courtesy of Kevin Hannavy.
Figure 3. Projection map of P-gp viewed from above the extracellular face of the membrane, showing a protein ring surrounding a large aqueous pore. Note the sixfold symmetry and the putative ‘gap’ in the protein ring providing access between the central pore and the lipid phase.
intensely with uranyl acetate and phosphotungstate. The pore is large, about 5 nm in diameter, and is the entrance to a cup-shaped chamber within the membrane, presumably formed by the twelve membranespanning a-helices ŽFigure 2.. An entry to this chamber from the lipid phase of the membrane is apparent, consistent with evidence that P-gp acts as a ‘flippase’ with drug substrates gaining access to their binding siteŽs. from the inner leaflet of the lipid bilayer.32,45,46 This chamber narrows as it passes through the membrane and is closed at the cytoplasmic face of the membrane, presumably by sequences from the nucleotide-binding domains and the cytoplasmic loops separating the transmembrane
Figure 4. Two possible orientations of the transmembrane helices forming the transmembrane pore of P-gp: the twofold symmetry model ŽA. or the mirror symmetry model ŽB..
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two domains interact directly. It is possible that direct interactions between the NBDs only occur in the presence of ATP andror substrate, and were therefore not apparent in the structure determined in the absence of these ligands. Alternatively, coupling between the NBDs may be mediated indirectly through interactions with the transmembrane domains. This is an important issue to resolve as it directly pertains to the mechanism of energy transduction. The structure of P-gp is very different from the low resolution structures determined for two other transporting ATPases, the Ca2q-ATPase and the HqATPase. In these two proteins the transmembrane a-helices are relatively tightly packed, rather than forming a large transmembrane pore. However, like P-gp, the complex which mediates protein translocation across the endoplasmic reticulum also has a 4]6-nm pore.52 The structure determined for P-gp is consistent with a general architecture for ABC transporters. The substrates transported by different ABC transporters can vary widely, from small ions to large polypeptides and polysaccharides:53 a common architecture with a large pore could readily be adapted to accommodate different sized substrates with relatively minor changes to the ‘gate’ at the cytoplasmic face of the membrane. Additionally, one ABC transporter ŽCFTR. is an ion channel and, again, the structure of P-gp is compatible with adaptation to such a function: indeed, studies on CFTR have led to the suggestion that several helices contribute to an aqueous pore gated at the cytoplasmic face of the membrane consistent with the structure determined for P-gp 54 ŽAkabas, M., personal communication.. Finally, it should be remembered that the structural data obtained for P-gp reflect a static conformation, yet the structure of P-gp is likely to be dynamic. It is generally accepted that P-gp undergoes significant conformational changes during its transport cycle, and ATP hydrolysis by the NBDs must induce conformational changes in the transmembrane domains to achieve active transport. Changes in crosslinking between cysteine residues introduced into P-gp imply a conformational change upon drug binding,35 and Fourier transform infrared spectroscopy shows that MgATP induces conformational changes involving a significant portion of P-gp. 23 These conformational changes do not involve a gross perturbation of structure, as the proportions of a-helix and b-sheet remain constant, and therefore presumably involve a movement of secondary structural elements or domains with respect to each other. Nevertheless,
the dynamic aspects of protein structure are essential for any understanding of its mechanism of action.
Prospects Although the direct determination of a structure for P-gp is an important step in understanding the mechanisms of transport, the resolution is low and much remains to be learned. Electron diffraction from large two-dimensional crystals is likely to im˚ although prove the resolution, perhaps to 6]8 A, atomic level resolution seems unlikely to be achieved in the foreseeable future. Nevertheless, low level resolution structural analysis together with careful mutagenesis and biophysical studies should give considerable insight into how this protein works, with consequent implications for developing improved approaches to reversing multidrug resistance in the clinic.
Acknowledgements We are grateful to the Cancer Research Campaign, Imperial Cancer Research Fund, and the Medical Research Council for support. We thank Kevin Hannavy for preparing Figure 2. CFH is a Howard Hughes International Research Scholar.
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