CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS

CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS CHAPTER 4 – STRUCTURE OF ABC TRANSPORTERS

65 STRUCTURE OF ABC TRANSPORTERS KENNETH J LINTON, MARK F ROSENBERG, IAN D KERR AND CHRISTOPHER F HIGGINS INTRODUCTION In order to understand the mechanism by which ABC transporters translocate solute across cellular membranes, structural data are essential Such data have been hard won This is primarily because of the difficulty inherent in overexpressing and purifying these proteins in an active form As for many membrane proteins, ABC transporters are often toxic to the cell or misfold when overproduced, and their vectorial active transport function is disrupted as they are purified Perhaps most importantly, the activity of many ABC transporters is influenced by their lipid membrane environment so ensuring that any purified protein is fully active, and therefore properly folded, is nontrivial There is also increasing evidence that the transmembrane domains (TMDs) of ABC CHAPTER transporters are highly flexible, a characteristic not conducive to ready crystallization Structural data have gradually emerged from a variety of approaches Many bacterial ABC transporters are multi-protein complexes with each of the four core domains encoded as a separate polypeptide (see introductory chapter to this volume) Several of the relatively hydrophilic nucleotide-binding domains (NBDs) have been overexpressed (including one of eukaryotic origin), purified and characterized at high resolution by X-ray crystallography (Table 4.1) Although such data tell us much about interactions with ATP, they have had little impact on our understanding of the mechanism of transport This is because binding of the translocated substrate is a property of the TMDs, and transport requires the interaction of all four domains Structural data for a complete transporter came TABLE 4.1 HIGH-RESOLUTION STRUCTURES OF NBDS NBD Organism Function Resolution (Å) Reference RbsA HisP MalK MJ0796 MJ1267 TAP1 Rad50 SMC E coli S typhimurium T litoralis M jannaschii M jannaschii H sapiens P furiosus T maritima Ribose uptake Histidine uptake Maltose uptake Unknown Amino acid transport Antigen presentation Double-strand DNA repair Double-strand DNA repair 2.5 1.5 1.9 2.7 1.6 2.4 2.5 3.1 Armstrong et al (1998) Hung et al (1998) Diederichs et al (2000) Yuan et al (2001) Karpowich et al (2001) Gaudet and Wiley (2001) Hopfner et al (2000) Lowe et al (2001) ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 66 ABC PROTEINS: FROM BACTERIA TO MAN initially from single-particle analyses and electron crystallography, and low- to mediumresolution structures are now available for three mammalian ABC transporters: the multidrug resistance P-glycoprotein (Pgp) to 25 Å by transmission electron microscopy (TEM) of single particles and to approximately 10 Å by electron cryomicroscopy (ECM) of two-dimensional (2-D) crystals (Rosenberg et al., 1997, 2001); MRP1 to 22 Å resolution by TEM of single particles and 2-D crystals (Rosenberg et al., 2001); and TAP to approximately 35 Å resolution (Velarde et al., 2001) The structure of YvcC from Bacillus subtilis has also been resolved to 25 Å by ECM The only X-ray crystallographic data for any complete ABC transporter came from a tour de force approach for the lipid A transporter (MsbA) from Escherichia coli (Chang and Roth, 2001; Chapter 7) In that study, over twenty E coli transporters were expressed and then tested under 96 000 crystallization conditions to yield an example that crystallized and diffracted (to a resolution of 4.5 Å) stabilizing its binding Among these are the expected interactions with the consecutive acidic amino acids at the C-terminus of the Walker B motif (D178 and E179 in HisP), and with the polar side-chains of the Walker A motif (K45, S46 and T47) The conserved polar amino acids of the Q-loop and the H-loop also make contact with ATP through water molecules bound within the ATP pocket One additional interaction is noteworthy: the adenine ring A Arm-II Arm-I STRUCTURES OF NBDS Although not the first NBD structure to be obtained (Stauffacher and colleagues solved the structure of the N-terminal NBD of E coli RbsA earlier; Armstrong et al., 1998), the HisP structure (Hung et al., 1998) was the first published description of an NBD at atomic resolution (1.5 Å) HisP, the NBD of the Salmonella histidine uptake system, is a single polypeptide domain of which two copies associate with the TMDs (HisQ and HisM) in the intact transporter (Kerppolla et al., 1991) The structure of HisP is shown in Figure 4.1A The most convenient description is that it comprises two ‘arms’ oriented approximately perpendicular to one another Arm-I contains an ABC-specific ␤-sheet subdomain (Karpowich et al., 2001), with the Walker A motif in a typical phosphate-binding loop conformation together with the Walker B motif (Walker et al., 1982) The perpendicular Arm-II, an ␣-helical subdomain, contains the ‘ABC signature’ motif The two other conserved motifs of ABC transporter NBDs, namely the Q-loop (Diederichs et al., 2000) and the H-loop (Linton and Higgins, 1998), are located at the interface of the two arms HisP was crystallized in the presence of ATP (Hung et al., 1998) The environment of the ATP molecule is depicted in Figure 4.1B A number of side-chains interact with nucleotide, B H211 K45 Y16 T47 S46 E179 Q100 D178 Figure 4.1 The structure of HisP and the environment of nucleotide within the ATP-binding pocket A, the two perpendicular arms of HisP are displayed in cartoon format with ribbons denoting ␣-helices and arrows representing ␤-sheets The bound ATP molecule is displayed in ball-and-stick format The colours represent: yellow, Walker A motif; red, Walker B motif; blue, H-loop; magenta, Q-loop; green, ABC signature B, The ATP molecule and side-chains of residues with which it interacts are displayed in ball-and-stick format Amino acid positions are indicated To ensure clarity, the backbone of the Walker A motif has been removed These and other structural diagrams were produced using the program Molscript (Kraulis, 1991) Carbon atoms are in gray (darker in the ATP molecule), oxygen atoms are red, nitrogen blue and phosphorus pink Reproduced with permission from Kerr (2002) STRUCTURE OF ABC TRANSPORTERS stacks against the side-chain of a well-conserved aromatic residue (Y16 in HisP) However, there are relatively few contacts on one side of the ATP molecule, and compared to other ATPases, the nucleotide appears somewhat exposed (Dreusicke et al., 1988); (Figure 4.1) that within these domains there is a degree of conformational flexibility between the subdomain motifs CONFORMATIONAL CHANGES IN THE NBDS The HisP structure (complexed with ATP) and the MJ1267 structure (complexed with Mg.ADP) have enabled comparison of NBDs in a pre-hydrolytic and post-hydrolytic state (Karpowich et al., 2001) These changes are illustrated in Figure 4.2 The ABC-specific subdomain (Arm-II) undergoes a 12–15° inward rotation, bringing it closer to Arm-I in the ATPbound structure compared with the ADP-bound structure In another allosteric membrane protein (the nicotinic acetylcholine receptor) a relatively small rotation at the presumed ligand-binding sites results in a substantial rotational movement in the channel-lining region (Unwin, 1993, 1995) Thus, although this 12–15 Å rotation may appear slight, its effects on the TMDs could be highly significant The rotation observed in MJ1267 appears to be centred on the Walker B motif, which is located close to the hinge between the two ‘arms’ The most dramatic change in orientation of residues involves the conserved glutamine of the Q-loop (Karpowich et al., 2001) In the ATP-bound conformation of HisP this amino acid is located within Å of the ␤-phosphate of ATP and interacts with it through a bound water molecule In stark contrast, the ADP-bound conformation sees this amino acid withdrawn from the nucleotide such that its closest approach is now Ͼ12 Å from the ␤-phosphate (Karpowich There are now structural data for NBDs in different conformational states (i.e nucleotidefree, or complexed with ADP or ATP) The most pertinent comparisons are made between different conformations of the same NBD Otherwise, to assess the conformational changes invoked by ATP binding, or by ATP hydrolysis followed by loss of Pi, NBDs from different ABC transporters must be compared This limits any analysis to regions conserved between NBDs Conformational change associated with ATP binding Rad50 provides the best model for comparison of nucleotide-free and ATP-bound conformations of NBDs This bacterial protein is involved in the repair of double-strand breaks in DNA and thus is an unusual paradigm for the interaction of NBDs in ABC transporters However, Rad50 contains sequences that unequivocally identify an ABC-transporter-like NBD, and the structure displays the characteristic L-shaped domain (Hopfner et al., 2000) Analysis of the ATP-bound and ATP-free forms of Rad50 indicates that there is a pronounced ordering of the Walker A, Walker B and Q-loop motifs upon interaction with nucleotide This suggests an induced fit of ATP This conclusion must be tempered with some caution as it is possible that the ATP-dependent dimerization of Rad50 might be responsible for this effect The implications of this dimer are discussed below However, analysis of the ADP-bound and ADPfree forms of another NBD protein (MJ1267) supports the hypothesis of induced nucleotide fit (see below) A second change observed in the ATP-bound form as compared with the ATPfree form of RAD50 is rotation of the ␣-helical subdomain relative to the ␤-subdomain Of course, it is difficult to assess whether rotation of the ␣-helical subdomain is a consequence of ATP binding or NBD:NBD dimerization However, evidence from other NBDs suggests Conformational change associated with ATP hydrolysis and release of Pi P P P A HisP:ATP P P A MJ1267:ADP Figure 4.2 The conformational changes associated with ATP hydrolysis Arm-II of the NBD undergoes a 12–15° outward rotation following hydrolysis of ATP and loss of phosphate The Q-loop (shown in black) is moved Å further from the bound nucleotide such that it can no longer interact with the ␤-phosphate Reproduced with permission from Kerr (2002) 67 68 ABC PROTEINS: FROM BACTERIA TO MAN et al., 2001) Thus, the Q-loop is a strong candidate for transmitting the conformational change associated with ATP hydrolysis to other domains of an ABC transporter In addition, the ABC signature motif (which is located in Arm-II) is displaced by about Å as a result of rotation of the ␣-helical subdomain and thus may also be involved in transmission of conformational change (Karpowich et al., 2001) Similar hinge-motions have been hypothesized in analyses of the structures of HisP (Hung et al., 1998), TAP1 (Gaudet and Wiley, 2001) and another Methanococcus NBD (MJ0796; Yuan et al., 2001) Whether these changes are the result of ATP hydrolysis or the release of phosphate from the post-hydrolytic intermediate remains unanswered Pharmacological studies indicate that conformational changes in the drug-binding site (TMDs) of Pgp occur upon phosphate release, rather than upon ATP hydrolysis (Martin et al., 2001; Rosenberg et al., 2001) The determination of NBD structures complexed with ADP.vanadate may confirm at which step the changes in NBD conformation occur Conformational change associated with release of ADP Conformational differences between the ADPbound and nucleotide-free structures of the NBD of the branched-chain amino acid transporter of Methanococcus jannaschii (MJ1267; Karpowich et al., 2001) may be seen as a model for the release of ADP from a post-hydrolytic NBD Studies of other ATPases suggest that the release of dinucleotide may often be accompanied by substantial conformational change (Scheirlinckx et al., 2001; Zhou and Adams, 1997) The two predominant changes are (i) a general destabilization of the NBD in the absence of nucleotide and (ii) alteration in the conformation of the H-loop The destabilization is reflected in higher crystallographic B-factors in several regions that provide interactions with the nucleotide The B-factor is a measure of the degree of flexibility in a region of a structure Thus, the Walker A, Walker B, H-loop and the ␤-strand containing the adenine ring-interacting aromatic residue (Tyr-16 in HisP) all exhibit higher B-factors in the absence of nucleotide, suggesting that release of ADP from the post-hydrolytic NBD is accompanied by a relaxation of the domain Put another way, it suggests an induced fit of nucleotide with NBD (Karpowich et al., 2001) The second structural effect is the change in conformation of the backbone of the conserved H-loop, which displaces the side-chains of the H-loop by as much as 12 Å This suggests that the H-loop may be involved in transmitting posthydrolytic conformational changes in an intact transporter No other changes of comparable magnitude are observed elsewhere (except in loops that are not conserved across the ABC transporter family) As previously stated, caution must be applied when comparing structural data on NBDs from different ABC transporters as crystal-packing forces may contribute to the conformational changes described However, it is particularly interesting that molecular dynamics simulations of NBDs in the presence or absence of nucleotide demonstrate both nucleotidedependent rotation of the ␣-helical subdomain and withdrawal of the Q-loop (Campbell and Sansom, personal communication) INTERACTION BETWEEN NBDS The structural data described above have been obtained for isolated, monomeric NBDs (in the case of RbsA, which contains two NBDs in a single polypeptide, only the first 259 amino acids corresponding to the N-terminal NBD were crystallized; Armstrong et al., 1998) Clearly, our understanding of the function and dynamics of ABC transporters would be greatly enhanced by a description of the structural and conformational interactions between domains Both NBDs in an ABC transporter are required for function (Azzaria et al., 1989; Gill et al., 1992) In the alternating catalytic cycle model only one ATP molecule is hydrolyzed at a time, with the ATPase activity alternating between the two NBDs (Hrycyna et al., 1999; Senior and Gadsby, 1997) Although we should not overlook the possibility that the two NBDs influence each other indirectly through their cognate TMDs, the simplest explanation is that they interact directly with each other In this respect, it is interesting that several of the monomeric NBDs have formed a crystallographic dimer (Kerr, 2002) These associations can be considered as hypothetical models for the interaction of NBDs in an intact ABC transporter Four alternative models have been presented for NBD:NBD association and are represented schematically in Figure 4.3 HisP forms a back-to-back crystallographic dimer in which STRUCTURE OF ABC TRANSPORTERS S A B S S S P P P P HisP MalK C D P P P S P S Rad50 ArsA Figure 4.3 Models for NBD association In each case ‘P’ refers to the location of the phosphate-binding loop (Walker A motif ), while ‘S’ represents the signature motif (absent from ArsA) A, HisP; B, MalK; C, Rad50; D, ArsA Reproduced with permission from Kerr (2002) the domains interact with each other through the exposed ␤-strands that constitute the ABC-specific ␤-sheet subdomain in Arm-I (Figure 4.3A) The perpendicular Arm-II and the ABC signature motif are proposed to interact with the TMDs, HisQ and HisM (Hung et al., 1998) The big drawback of this interface is the very small surface area buried by dimer formation (about 1000 Å2) Detailed comparison of dimer interfaces in proteins suggests such a small buried surface area may be the result of crystal-packing forces, rather than a physiologically relevant dimer (Kerr, 2002) The NBDs of the thermophilic maltose transporter (MalK) interlock in the crystal structure as shown in Figure 4.3B, with close contacts between the hinge regions of the two NBDs In particular, the Q-loops are in close contact across the dimer interface (4 Å), consistent with a role in transmission of inter-domain conformational change In this dimer, the TMDs would be in close apposition to the ABC signature motifs (Diederichs et al., 2000) For Rad50, the crystallographic dimer (Figure 4.3C) shows the two monomers interact in a head-to-tail fashion Interestingly, nucleotide binds at the NBD:NBD interface in Rad50 and is coordinated by interactions with the Walker A and B motifs of one NBD, and the ABC signature motif of the other NBD This orientation provides extra stability for the nucleotide and provides a possible explanation for domain:domain interaction upon ATP hydrolysis through the ABC signature motifs (Hopfner et al., 2000) A further potential dimerinterface model is derived from the structure of ArsA (Figure 4.3D), the ATP-hydrolytic domain of the bacterial arsenic transporter (Zhou et al., 2000) Although ArsA is not a member of the ABC transporter family (as it does not possess the characteristic ABC signature motif), it may be informative since the ArsA dimer is formed from a single polypeptide with two ATP-binding sites Thus, the association of the two domains cannot be an artifact of crystallization conditions In this structure the two ATP-binding pockets are considerably closer together (about 15 Å) than in the other three models (Zhou et al., 2000) In attempting to assess the validity of the four dimer interface models, a number of considerations are necessary First, what biochemical evidence supports the association state? Second, are there theoretical considerations that may have an impact? Third, does the interface between the NBD and the TMD, as suggested crystallographically for MsbA (Chang and Roth, 2001), rule out any of the proposed models? The biochemical data are diverse and consist of attempts to measure direct interactions (e.g by crosslinking of cysteine residues), as well as indirect interactions (e.g by examining the effects 69 70 ABC PROTEINS: FROM BACTERIA TO MAN that mutations in NBDs have on the transport complex) Data for MalK suggest that mutation of residues proximal to the Q-loop to cysteine can result in dimerization of MalK, consistent with the close apposition of these loops in the MalK dimer model (Hunke et al., 2000) Cysteine crosslinking data for Pgp suggest that the two Walker A motifs may be as close as 15–20 Å apart (Loo and Clarke, 2000; Urbatsch et al., 2001) This appears to be consistent only with the ArsA structure, as these motifs are more than 25 Å apart in the Rad50, HisP and MalK dimer models However, fluorescence resonance experiments on Pgp suggest that the distance between Walker A motifs might be 30–35 Å, which would be satisfied by models other than ArsA (Qu and Sharom, 2001) A considerable amount of data has been obtained for bacterial ABC transporters that is rather more indirect, in that it pertains more to the interaction of NBDs with the TMDs However, the nature of this interaction could clearly provide a considerable constraint on potential NBD:NBD interactions For the histidine and maltose transporters, the interaction of the TMDs with the NBDs has been assessed by mutagenesis and co-purification studies (Liu et al., 1999; Petronilli and Ames, 1991) Mutations on the face of HisP that is proposed to interact with the TMDs (i.e the upper surface of Arm-II in Figure 4.1A) diminished copurification with the TMDs, suggesting that the association of the domains is disrupted Similarly, for the maltose transporter, mutations in the conserved region linking TM ␣-helices and of MalF and MalG (containing the so-called ‘EAA’ motif) that disrupted function could be rescued by ‘suppressor’ mutations in the ␣-helical ABC-specific domain (Arm-II; Mourez et al., 1997) Some residues within the EAA motif could also be crosslinked to three residues in the same ␣-helix of the ␣-helical subdomain of MalK (Hunke et al., 2000) Again this helix is on the upper face of Arm-II as viewed in Figure 4.1A The main caveat is that the results can also be explained by allostery, i.e that the mutations leading to a loosening of the HisQMP2 or MalGFK2 complexes are not necessarily at the domain:domain interfaces but are downstream effects of these mutations The interaction of nucleotide with NBD proteins has been cited as support for the Rad50 dimer model (Hopfner et al., 2000; Karpowich et al., 2001) Rad50 in the absence of nucleotide is a monomer in solution and in the crystal However, ATP (or indeed the non-hydrolyzable analogue AMP-PNP) leads to dimerization of Rad50 in solution and in the crystal Mutations in the ABC signature motif disrupt ATPdependent dimerization in Rad50 (Hopfner et al., 2000) However, several studies have indicated that ATP does not promote the dimerization of other NBDs (see Kerr, 2002) Furthermore, SMC, another DNA-interacting protein with an ABC-transporter-like NBD, does not show the interlocking-L arrangement that characterizes Rad50 Instead, SMC adopts a hexameric association in the crystal irrespective of the presence of nucleotide (Lowe et al., 2001) Recently, a structure has been obtained at 4.5 Å resolution for the entire prokaryotic lipid A transporter MsbA (Chang and Roth, 2001) This protein is a ‘half transporter’, containing a single NBD and a single TMD within the same polypeptide Although a considerable proportion of the NBD is not resolved in the crystal structure, the structural data illustrate the interface between the TMD and the NBD, and so constrain putative NBD dimer interfaces (see above) The most noticeable feature of the MsbA structure (Figure 4.4; a complete description of the structure is given in Chapter of this volume) is the recognition of intracellular subdomains (ICDs) linking the TMD and NBD In the orientation shown (Figure 4.4) the sequences in the NBD that interact with the ICDs are clearly visible (purple) Three conserved motifs of the NBDs which pack against the ICDs are the Walker B ␤-strand, the Q-loop and the first ␣-helix of the ␣-helical subdomain All three can be mapped onto the lower surface of Arm-II in HisP (Figure 4.1A) The implication of this admittedly incomplete structure is that the MalK dimer interface (in which the Q-loops are in very close apposition; Diederichs et al., 2000) is not a feasible model for an NBD:NBD dimer interface in intact ABC transporters How the crosslinking data for MalK (see above), which suggest that the upper face of the ␣-helical subdomain is in contact with the TMDs, can be reconciled is also unclear In conclusion, the current data seem to favour the orientation of the two ATP-hydrolytic domains of Rad50 (and possibly ArsA) as a model for NBD:NBD interactions within an intact ABC transporter It is worth pointing out that the published MsbA structure consists of two molecules tilted together at the extracellular face and splayed apart at the intracellular side, separating their NBDs such that they not share an interface (Chang and Roth, 2001) Thus, perhaps the NBDs not interact or STRUCTURE OF ABC TRANSPORTERS Figure 4.4 Domain:domain interactions in MsbA The structure of a single MsbA molecule is displayed in a cartoon format The ␣-helices of the TMDs are displayed in blue, while their intracellular extensions which comprise the ICD are colored green The NBD is predominantly colored yellow, but the three regions which interact with the ICDs are colored purple These comprise the Q-loop and Walker B motif and the first ␣-helix of Arm-II interact only transiently during the transport cycle Progress towards a higher-resolution structure of mammalian ABC transporters will be required to resolve this issue STRUCTURES OF INTACT ABC TRANSPORTERS Pgp is the best-characterized mammalian ABC transporter and this section is focused on that protein Data obtained for other mammalian ABC transporters are broadly consistent and, where appropriate, are compared and contrasted The MsbA structure is described in detail elsewhere in this volume (Chapter 7): a comparison with Pgp illustrates unresolved questions Structure determination requires not only pure protein, but a reasonable degree of confidence that the purified protein has retained its native fold This poses a significant problem when working with large molecular weight membrane proteins Pgp activity is dependent on the lipid environment of the membrane (Callaghan et al., 1997), while the first step in purification requires disruption of the lipid bilayer and solubilization of the protein components using detergent Not surprisingly, this destroys measurable activity of Pgp To demonstrate that the solubilized and purified protein has retained, or can regain, the native protein fold, the detergent must be replaced by lipid so that activity can, once again, be measured The choice of detergent is therefore crucial to the success of the purification process The detergent must solubilize the membrane protein without irreversible denaturation and it must be possible to replace the solubilizing detergent with lipids The non-ionic detergent, dodecyl␤-D-maltoside has the requisite characteristics and has proved invaluable for solubilization of Pgp from multidrug resistant Chinese hamster ovary cells (Callaghan et al., 1997) A LOW-RESOLUTION STRUCTURE FOR PGP A low-resolution (25 Å) structure of active Pgp (shown by drug-binding and drug-stimulated ATPase activity) was determined by TEM of single particles In this technique, multiple images of single particles with a similar orientation were aligned and averaged to produce an image of higher signal:noise ratio (Rosenberg et al., 1997) Single particle analysis was initially carried out on reconstituted Pgp because, compared with solubilized protein, the lipid bilayer confines all the particles in the z axis (i.e in the plane of the membrane) Thus, the molecules only exhibit rotational freedom in the x, y dimensions, limiting the potential orientations that they can adopt Furthermore, when reconstituted into a lipid environment, Pgp was shown to exhibit drug binding and ATPase activity similar to that of the protein in its native membranes The reconstituted protein was examined under negative-stain, such that only the stain-accessible surface of the molecule was observed More than 70% of the protein reconstituted into the lipid bilayer adopted a single orientation TEM of this material (Figure 4.5A) projected an electron-dense ring of protein 12 nm in diameter with both twofold and sixfold symmetry surrounding a central chamber of approximately nm diameter The twofold symmetry in this structure was consistent with 71 72 ABC PROTEINS: FROM BACTERIA TO MAN A B C D Figure 4.5 Projection maps of Pgp Images derived from single particle alignment and averaging of negatively stained Pgp particles, using contours and shading to delineate stain (black) and protein (white) boundaries A, averaged projection map of Pgp reconstituted into proteoliposomes B, C and D, averaged projection maps of solubilized Pgp particles Three classes of particle were observed, which differ in their orientations with respect to the electron beam B, face-on projection of the extracellular face of Pgp C, face-on projection of the cytoplasmic face of Pgp D, side-on view of Pgp; the two NBDs are indicated Reproduced with permission from Rosenberg et al (1997) the presence of two homologous TMDs and the sixfold symmetry is consistent with six pairs of transmembrane ␣-helices, each pair linked by an extracellular loop To obtain images of different surfaces, the particles were examined as detergent solubilized material This provided several different views of the particles (Figure 4.5B, C and D) One projection (Figure 4.5B) closely resembled that of the reconstituted protein (Figure 4.5A) Preferential labeling of this surface of solubilized Pgp particles by lectin-gold shows it to be glycosylated and therefore consistent with a view of the extracellular surface of the TMDs of Pgp Because the chamber accumulated the uranyl acetate stain it is likely to be aqueous and thus open to the extracellular milieu Futhermore, at least for this surface, the protein has a similar fold in the lipid bilayer and when solubilized in detergent A second projection (Figure 4.5C) was also circular and 10–12 nm in diameter However, this surface, with no central chamber and two nm diameter lobes, was distinct from the extracellular view These lobes are an appropriate size for the 200 amino acid NBDs The third projection (Figure 4.5D) was asymmetric in shape, with three small lobes on one half of the particle and two larger lobes on the other half This projection probably represents a side view, in which the two larger lobes correspond to the two NBDs and the three small lobes correspond to the TMDs (the hexagonal symmetry of the TMDs when viewed from above would be expected to project three electron-dense lobes when viewed from the side) Thus, the shape of the Pgp particle approximates a short and fat cylinder, 10 nm in diameter and about nm high The lipid bilayer is about nm in thickness, suggesting that about one-half of the molecule resides within the membrane The TMDs form a chamber in the membrane, open at the extracellular face The chamber is closed at the cytoplasmic face of the membrane and the two nm lobes probably correspond to the NBDs MEDIUM-RESOLUTION STRUCTURE OF P-GLYCOPROTEIN Single particle analysis and negative stain limit the resolution of the data To obtain higher resolution data 2-D crystals of Pgp were obtained and imaged using low-dose electron cryomicroscopy (ECM) Precipitant-induced, large, well-ordered 2-D crystals of Pgp can be grown reproducibly at the air/water interface of a droplet Projection images of frozen-hydrated, 2-D crystals displayed reflections to approximately Å resolution (Rosenberg et al., 2001) Importantly, because crystallized protein cannot be analyzed for function, the unit cell of the crystals was very similar to the size and shape of the single particles, making it likely that the native protein fold had been preserved Tantalizingly, the resolution of the processed image remains around 10 Å, just outside that required for recognition of secondary structural features ECM of the holoenzyme, unlike negative stain, produced a 2-D projection of all electron densities in the 3-D protein (Figure 4.6) The STRUCTURE OF ABC TRANSPORTERS a b 10 Å Figure 4.6 Projection map of Pgp determined by electron cryomicroscopy at 10 Å resolution Solid lines indicate density above the mean Twelve major densities (A–F with their pseudosymmetric densities A؅–F؅ ) are related by a pseudo-twofold symmetry axis centered at the star A region of low protein density corresponds to an aqueous chamber within the membrane The areas circled in blue at opposite ends of the molecule probably include densities corresponding to the NBDs; the three-dimensional reconstruction shows they are at the cytoplasmic face of the membrane Reproduced with permission from Rosenberg et al (2001) projection structure approximates to an elliptical ring of 91 Å ϫ 60 Å with a slightly asymmetric, central low-density region entirely consistent with the surface views obtained by analysis of single particles in negative stain As expected, the protein has distinct pseudotwofold symmetry with several pairs of clearly related density peaks (Figure 4.6) The consistency with the low-resolution structure is more easily recognized in a 3-D map of the protein, generated from negatively stained 2-D crystals by imaging the crystal lattice from different angles Although at lower resolution, this analysis provided information about the spatial organization of the four domains Sections through the 3-D map at approximately 10 Å intervals through the plane of the membrane are shown in Figure 4.7 Working up from what is thought to represent the intracellular surface of the transporter, two large densities (see filled arrows in Figure 4.7A) are related by twofold pseudosymmetry These domains presumably reflect the NBDs and are of an appropriate size to each accommodate HisP (see open arrow, Figure 4.7A) In the next section through the transporter (Figure 4.7B) the NBDs are still apparent (indicated by arrows) but there are now two extra electron densities either from a second lobe of each of the NBDs, or from intracellular loops of the TMDs (or, possibly, a combination of both) The four lobes now surround a distinct centre of low electron density indicating that the central chamber of the transporter extends deep into the plane of the membrane At this resolution it is not possible to ascertain whether the specific residues which block the chamber come from the cytoplasmic loops of the TMDs or from the NBDs (although no evidence for such a role for the NBDs could be found by in vivo labeling studies; Blott et al., 1999) Towards the midpoint of the membrane, two arcuate domains form almost a complete ring of protein around the central chamber Each domain has three higher electron densities, which presumably correspond to pairwise clustering of the six putative transmembrane ␣-helices of each TMD (labeled 1–3 and 4–6 in Figure 4.7D) There are noticeable ‘gaps’ between the two TMDs within the plane of the membrane, potentially permitting side-access to the chamber from the lipid phase (arrows in Figure 4.7D) At the extracellular surface of the transporter (Figure 4.7E) the electron densities probably include contributions from both the TM ␣-helices and the extracellular loops and a pronounced gap in the protein ring is evident Comparison of Pgp with low-resolution structures of MRP1 and TAP Structures for MRP1 and TAP have been resolved to 22 Å resolution (Rosenberg et al., 2001) and around 35 Å (Velarde et al., 2001), respectively MRP1, TAP and Pgp are members of different subfamilies of ABC transporters MRP1 has an extra TMD (TMD0) in addition to the four core domains, and TAP is a heterodimer of two ‘half ABC transporters’, TAP1 and TAP2 The primary sequence of the TMDs of TAP is particularly different from those of Pgp and MRP1, and probably reflects the specialized nature of TAP for antigen presentation to MHC class I molecules Despite these differences, single particle images of MRP1 and TAP 73 74 ABC PROTEINS: FROM BACTERIA TO MAN B A C h k E D F Outside E D C B A * TM TM Lipid bilayer NBD NBD Inside Chamber Figure 4.7 3-D map of Pgp Panels A to E represent slices in the plane of the crystal (x, y) (parallel to the plane of the membrane), with each slice having an approximate thickness of 10 Å and progressively moving from the intracellular (A) to the extracellular (E) side of the membrane The scale bar ‫ ؍‬28 nm In the inset of panel A, two HisP monomers (Hung et al., 1998) to the same scale are modeled onto the structure as ribbon diagrams Panel F shows two sketched views of Pgp with the TMDs in red and the NBDs in blue The upper panel shows a cross-sectional sketch of the Pgp molecule indicating the approximate plane of sections The lower panel shows a view of Pgp in the plane of the membrane as viewed from the extracellular face Reproduced with permission from Rosenberg et al (2001) are similar in size and shape to those for Pgp Each structure consists of a ring of protein surrounding a large central hydrophilic chamber, which is open at one side of the membrane and closed at the other with two large protein lobes In the MRP1 and TAP structures it is not possible to say whether the ‘open’ side is equivalent to the intra- or extracellular face of the membrane, although it is expected to be extracellular by virtue of comparison with Pgp and because the two large lobes are probably the intracellular NBDs Interestingly, analysis of TAP2 by itself suggests that the TMD of TAP2 can form an arcuate structure independently of TAP1, consistent with the interpretation that each TMD contributes half of the chamber in the membrane (as also seen for the MsbA X-ray structure; see below) In single particles of MRP1, a particularly large protein density at the outer side of the ring may represent the additional TMD0 Although MRP1 crystallizes as a dimer, at current resolution it is not possible to demonstrate that the crystallization dimer is ‘double barreled’ with a separate chamber per molecule, although this would seem likely given the structure of single particles of MRP1 Comparison of Pgp with the structures of MsbA and YvcC MsbA is a ‘half transporter’ with one NBD and one TMD It is expected to function as a homodimer to transport lipid A across the inner membrane of E coli The higher-resolution structure (4.5 Å) for MsbA represents an important advance (Chang and Roth, 2001; Chapter 7) For the first time it is definitively demonstrated that STRUCTURE OF ABC TRANSPORTERS TMDs of ABC transporters cross the membrane via ␣-helices (six in the case of MsbA) The interdigitation of intracellular loops of the TMD and structural elements of the NBD define this interdomain interface and suggest a mechanism for signal and energy transduction between domains In MsbA the two TMDs form a block of ␣-helices angled towards each other at approximately 45° to the perceived plane of the membrane Only towards the outer leaflet of the membrane the two TMDs of the homodimer interact, forming a structure that resembles a pitched roof with the apex towards the extracellular surface of the membrane and the chamber below Extending this analogy, the gable ends are missing from the structure, leaving large gaps between the TMDs towards the inner leaflet of the membrane (Similar, but much less pronounced gaps are evident in the Pgp structure and may represent a route for substrate to access the center of the chamber directly from the lipid phase.) The MsbA chamber is therefore closed at the extracellular surface and open at the intracellular surface, in stark contrast to the mammalian examples At the cytoplasmic face of the membrane, the NBDs of MsbA are distinct from each other and not appear to interact YvcC is also a bacterial ‘half transporter’, homologous with MsbA, LmrA and both halves of Pgp YvcC purifies as a monomer in detergent micelles and forms higher-order structures when the detergent is removed These higher-order structures have been analyzed by ECM and resolved to 25 Å The multimers form rather beautiful ring shapes, 40 nm in diameter, comprising 48 monomers of YvcC These are arranged in two layers of 12 dimers Of particular interest here are the three dimer interfaces within the particle Two of these interfaces resemble the arrangement found in the MsbA dimer and form a chamber, closed at the extracellular surface The third arrangement appears to share a large interface between the NBDs and would form a chamber open at the extracellular surface In comparison with the lower-resolution structures of mammalian ABC transporters the MsbA structure is both similar and different From all structures, it is clear that a chamber is formed in the membrane from two ‘half transporters’ and that gaps between the two TMDs could provide access to the lipid phase within the plane of the membrane The major difference between the structures is inherent in the angle at which the transmembrane ␣-helices cross the membrane and this dictates the open- ing of the chamber either to the cytoplasmic side of the membrane (as in MsbA) or to the extracellular side of the membrane (as in Pgp) How to reconcile this difference? YvcC does not help as different dimer interfaces within the particles are consistent with either structure In the absence of data demonstrating function of the purified MsbA and YvcC, it is difficult to be confident that this material represents the native protein fold or that the physiological dimer interface has survived the purification protocol (YvcC solubilized from the membrane is monomeric) It is worth remembering that crystals grown from isolated NBDs have reported a number of different dimer interfaces, not all of which can be correct (see above) Furthermore, the arangement of the transmembrane ␣-helices in MsbA is inconsistent with in vivo crosslinking data for the TMDs of Pgp (DRS, KJL and CFH, unpublished data; Loo and Clarke, 1996b), although crosslinking data can be misleading Alternatively, flexibility is a prerequisite for vectorial transport and Pgp is demonstrably very flexible (see below) and therefore the apparent differences could reflect different conformational states Additional data are required before it will be possible to fully interpret structural and biochemical data obtained to date Comparison of the structure of ABC transporters with other transporters The overall structure of ABC transporters appears different from that of the P-type iontransporting ATPases Principally, the large chamber in the membrane formed by the TMDs of Pgp contrasts with the relatively tight packing of the transmembrane ␣-helices of the iontranslocating ATPases (Toyoshima et al., 2000) The structure of NhaA, a secondary transporter mediating Hϩ/Naϩ antiport, has been determined by similar methods (Williams et al., 1999), as has the bacterial multidrug transporter EmrE (Tate et al., 2001) Again, in contrast to Pgp the membrane-spanning segments of these proteins are packed into a relatively asymmetric tight bundle, which lacks a large central pore The reason for these differences is unclear, but may be related to the nature of the substrates transported Individual ABC transporters are often very specific for a given substrate (Pgp is unusual in this respect), but when different members of the superfamily are considered it seems clear that a similar domain architecture 75 76 ABC PROTEINS: FROM BACTERIA TO MAN is capable of handling a wide diversity of substrates Cations, amino acids, large polypeptides and phospholipids are all substrates of ABC transporters and a chamber in the membrane may provide a universal architecture which is more adaptable to different-sized substrates than closely packed ␣-helices Interestingly, in the ER membrane the translocon for general protein export also has a central chamber of similar dimensions to that of Pgp (Hamman et al., 1997) Monomer or dimer? The quaternary organization of Pgp and other ABC transporters has long been a subject of debate There are biochemical data in support of both monomeric (Loo and Clarke, 1996a; Taylor et al., 2001) and multimeric (Boscoboinik et al., 1990; Naito and Tsuruo, 1992; Poruchynsky and Ling, 1994) Pgp Structural studies strongly suggest the Pgp particle and unit cell is a monomer (Rosenberg et al., 2001) Lectin-gold labeling also suggests that the unit cell of the crystal lattice and the single particles are composed of monomeric Pgp (Rosenberg et al., 1997) Lectin recognizes the sugar residues added to the first extracellular loop of Pgp When single particles of Pgp were labeled with lectin-gold and analyzed by TEM only one gold particle was observed per Pgp particle, consistent with only one copy of the first extracellular loop per Pgp particle (or unit cell) Thus, we can be reasonably certain that the structural unit of Pgp is a monomer with 12 membrane-spanning ␣-helices forming the pathway through the membrane Is this the functional unit? Evidence that monomeric Pgp, observed by single particle analysis, binds drugs and has a drug-stimulated ATPase activity not substantially different from native Pgp (Callaghan et al., 1997; Rosenberg et al., 1997) strongly supports this view However, perhaps the clearest evidence comes from genetic analysis of LmrA, a bacterial homologue of Pgp (van Veen et al., 2000) LmrA is a half molecule of Pgp with one NBD and one TMD Dominant-negative mutant and wild-type LmrA molecules were purified, mixed in different ratios, reconstituted into proteoliposomes, and tested for transport activity The level of activity varied with the ratio of wild-type to mutant forms in a manner consistent only with a model in which two monomers of LmrA form a functional homodimer, equivalent in domain structure to a monomer of Pgp In contrast to Pgp, MRP1 crystallizes as a homodimer, although the functional relevance of this in biological membranes is unknown and single particles of the protein are monomeric (see above) Recent studies on CFTR suggest that dimerization mediated by a cytoplasmic accessory protein (CAP70) can lead to the formation of a double-barreled channel complex, which can alter the regulation of the channel (Wang et al., 2000) but is not necessary for function (Ramjeesingh et al., 2001) Thus, although the activity of Pgp and other ABC transporters may be regulated by dimerization (transient or otherwise), the available evidence suggests that the minimal functional unit is a monomer, and that dimers would have two aqueous chambers each formed by 10 to 12 TM ␣-helices CONFORMATIONAL CHANGES IN THE TMDS OF PGP DURING TRANSPORT It is clear from biochemical studies of Pgp that the protein is very flexible within the membrane and that the transport cycle involves changes in conformation (Chapter 6) Such changes have now been seen directly (Rosenberg et al., 2001) Pgp function can be blocked at different stages of the ATP catalytic cycle by the non-hydrolyzable ATP analogue AMP-PNP, or trapping ADP in the NBDs using vanadate (Senior and Gadsby, 1997) Projection maps of the extracellular surface of the protein in negative stain showed that the TMDs of Pgp adopt significantly different conformations depending on the occupancy of the NBDs (Figure 4.8) Such conformational changes were predicted by biochemical data (Chapter 6) but their magnitude is unexpected In the presence of AMP-PNP, which mimics the ATP-bound form of Pgp, the overall shape was roughly triangular with three distinct protein densities surrounding the central pore In the presence of ADP and vanadate, Pgp was trapped in an intermediate catalytic conformation Thus, three distinct conformations of Pgp correspond to the nucleotide-free, nucleotidebound, and post-hydrolytic (vanadate-trapped) steps of the catalytic cycle These can be combined with biochemical data to generate a model for Pgp transport (Figure 4.8) For clarity, the ATPase cycle for only one NBD is shown, although both NBDs are known to be required for function ATP binding to Pgp generates the first major conformational change ATP binding has also been shown to reduce significantly the affinity of Pgp for vinblastine (Martin et al., STRUCTURE OF ABC TRANSPORTERS [Substrate]in ATP S Pgp Step I Pgp Step II Step IV [Substrate]out Pgp ADP ATP Step III Pi Pgp ADP.Pi Figure 4.8 Conformational changes during the transport cycle of Pgp The model is based on data presented here and published elsewhere (Senior and Gadsby, 1997) For clarity the ATPase cycle for one NBD is shown, although in Pgp both NBDs are required and operate in an alternating catalytic cycle Pgp in its native state (the absence of bound nucleotide) binds drug substrate from the inner leaflet of the lipid bilayer at the intracellular face of the membrane (step I) Subsequently, ATP is bound by the NBD(s) (step II), inducing a conformational change which results in a reduction in the affinity of the drug-binding site and its reorientation such that it is exposed to the extracellular milieu (probably the aqueous phase in the chamber formed by the TMDs of Pgp) ATP hydrolysis via at least one intermediate (steps III and IV) returns the protein to its starting configuration Reproduced with permission from Rosenberg et al (2001) 2001; Rosenberg et al., 2001) Thus, vinblastine binding to its high-affinity site must precede ATP binding in the transport cycle (Figure 4.8, step I) Upon binding ATP, a major conformational change is induced in the TMDs, which reduces the affinity of the vinblastine-binding site, and hence permits release of drug (Figure 4.8, step II) Presumably, this conformational change also reorientates the drug-binding site so that it is exposed extracellularly This most probably involves release of drug into the aqueous chamber; Pgp and its bacterial homologue LmrA are known to bind drug directly from the inner leaflet of the membrane (Bolhuis et al., 1996; Shapiro and Ling, 1997) and for maximal efficiency they would be expected to release drug directly to the extracellular environment Thus, any drug re-entering the cell would have to diffuse across the phosphate head groups of the outer leaflet and flip to the lipid chains of the inner leaflet, where it would again be accessible to Pgp Consistent with this idea, it has been shown for the bacterial drug transporter LmrA that ATP binding results in reorientation of the drug-binding site such that it is exposed to the extracellular rather than intracellular face of the membrane (van Veen et al., 2000) It is therefore likely that the major conformational change following ATP binding both reduces affinity and reorientates the drug-binding site Thus, ATP binding, rather than hydrolysis, appears to provide the energy for drug translocation, although this is still a controversial issue Following ATP hydrolysis, but prior to release of ADP or Pi (the vanadate-trapped state), there is a further conformational change (Figure 4.8, step III), although the drug-binding site remains low affinity Only on release of ADP and/or Pi (Figure 4.8, step IV) does the protein return to the original configuration with high-affinity drug binding, and so is ‘reset’ to enter another transport cycle (Sauna and Ambudkar, 2001) CONCLUSIONS We can be confident of the two-arm, L-shaped structure of the NBDs of ABC transporters The recent MsbA structure has provided important information as to how the NBD interacts with its cognate TMD through so-called ICDs consisting of intracellular loops of the TMD However, to fully understand transport, it is necessary to understand domain–domain interactions: the availability of structural data for full-length ABC transporters is still limited Nevertheless, data now appearing provide intriguing insights, guide the design of new experiments, and 77 78 ABC PROTEINS: FROM BACTERIA TO MAN demonstrate that technical problems can be overcome Transport is, by definition, a vectorial process, which is likely to require conformational changes, and thus single structures will never tell the whole story This is evidenced by the major rearrangement of transmembrane domains seen in P-glycoprotein during the transport cycle Only a combination of structures obtained under a variety of conditions, for example with and without substrate and/or ATP, together with biochemical data will give a complete insight into precisely how transmembrane transport is achieved Recently the structure of another bacterial ABC transporter, BtuCD, has been published (Locher et al., 2002) The structure differs substantially from the MsbA structure in a number of respects The NBDs (BtuD) form a dimer interface similar to that of Rad50, with the signature motif of one NBD in a position to co-ordinate with ATP bound to the other NBD The TMDs (BtuC) interact to form a small chamber in the membrane open to the extracellular melieu, which the authors plausibly suggest could accommodate the substrate, vitamin B12 There are no real 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STRUCTURE OF ABC TRANSPORTERS TMDs of ABC transporters cross the membrane via ␣-helices (six in the case of MsbA) The interdigitation of intracellular loops of the TMD and structural elements of the... above) The most noticeable feature of the MsbA structure (Figure 4. 4; a complete description of the structure is given in Chapter of this volume) is the recognition of intracellular subdomains (ICDs)