Physiology 22: 122-130, 2007;
doi:10.1152/physiol.00046.2006 
1548-9213/07 $8.00
Physiology, Vol. 22, No. 2, 122-130,
April 2007
© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.
REVIEW
Structure and Function of ABC Transporters
Kenneth J. Linton
MRC Clinical Sciences Centre, Imperial College Hammersmith Hospital Campus, London, United Kingdom, kenneth.linton{at}csc.mrc.ac.uk
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Abstract
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ATP binding cassette transporters are ubiquitous integral membrane proteins that actively transport ligands across biological membranes, a process critical for most aspects of cell physiology. These proteins are important clinically and economically. Their dysfunction underlies a number of human genetic diseases, and the ability of some to pump cytotoxic molecules from cells confers resistance to antibiotics, herbicides, and chemotherapeutic drugs. Recent structure analyses interpreted in light of a large body of biochemistry has resulted in the ATP-switch model for function in which the paired nucleotide binding domains switch between an ATP-dependent closed conformation and a nucleotide-free, open conformation to drive the translocation of ligand.
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ABC Transporters are Ubiquitous and Clinically Important
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ATP binding cassette (ABC) transporters are found in all species and many species have lots. There are, for example, 28 in Saccharomyces,58 in Caenorhabditis, 51 in Drosophila,129 in Arabadopsis,and the 69 ABC transporters in E. coli account for almost 5% of its genomic coding capacity (27). Typically, they transport ligands across cellular lipid membranes, which is critical for most aspects of cell physiology, including the uptake of nutrients and elimination of waste products, energy generation, and cell signalling.
There are 48 ABC transporters in humans and mutations in many are at the root of genetic disorders including a bleeding disorder (2) and a number of eye (39) and liver diseases (20), all of which are caused by the failure to export a specific ligand across a lipid bilayer (Table 1
; Refs. 10, 14). The normal function of some human ABC transporters is to secrete from cells cytotoxic compounds (dietary cytotoxics and therapeutic drugs). These transporters (P-glycoprotein, BCRP, and MRP1) are highly expressed in the gut, liver and kidneys where they restrict the bioavailability of administered drugs. P-glycoprotein and BCRP in particular are also expressed in the epithelia of sensitive tissues [for example, the brain and placenta (22, 50)] and in stem cells (66), where they perform a barrier function (48). There are also a few atypical ABC proteins. In this category, I have included the cystic fibrosis transmembrane regulator (CFTR) and sulphonyl urea receptor (SUR) because, although they have the requisite domains to ostensibly function as transporters (see below), CFTR is a channel for chloride ions (55) and SUR is a regulator of a potassium channel to which it is directly coupled in a hetero-octameric complex (19). These proteins share this category with others that are derived from the energy-converting domains, the nucleotide binding domains (NBDs), but which have evolved to couple the energy released by binding and hydrolysis of ATP to processes other than transbilayer transport. These include proteins involved in chromatin organization (13), DNA repair, telomere maintenance (15, 44), and mRNA trafficking through the nuclear pore (23). The molecular mechanism described below for ATP catalysis by the NBDs and its coupling to transport is also likely to describe the conformational changes involved in these processes.
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ABC Transporters Have Four Core Domains
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To achieve export [import appears to require an additional periplasmic binding domain (PBP)], ABC transporters require a minimum of four domains (FIGURE 1). Two transmembrane domains (TMDs) form the ligand binding sites and provide specificity, and two NBDs bind and hydrolyze ATP to drive the translocation of the bound ligand. The NBDs, but not the TMDs, are homologous throughout the family and have several characteristic motifs including the Walker A and B motifs common to many nucleotide binding proteins and others like the ABC signature, stacking aromatic D, H, and Q loops, which are unique to the family. The four domains can be encoded on different polypeptides, as in the vitamin B12 importer BtuCD from E. coli,or can be fused together in any combination (27), including as a single four-domain protein such as found in P-glycoprotein.
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FIGURE 1. The minimal ABC transporter has four domains
Two transmembrane domains (TMDs) bind ligand, and transport is driven by ATP binding and hydrolysis by the two nucleotide binding domains (NBDs). The TMDs from different subfamilies of ABC transporters are not necessarily homologous. The NBDs are homologous throughout the family. Each NBD has seven highly conserved, but not invariant, motifs.
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The ATP Catalytic Cycle
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A considerable body of biochemical and genetic studies has been amassed on many different ABC transporters from different species. Depending on the ligand, the host organism, and also protein function (CFTR, for example, allows careful kinetic study of how the function of the NBDs controls the opening and closing of the chloride channel formed by the TMDs), different systems have been able to provide distinct insights into how these proteins work. Mammalian cells can be selected for overexpression of P-glycoprotein by cultivation in the presence of cytotoxic drugs. This facilitated purification of the protein and also characterization of its ATP catalytic cycle more than 10 years ago (51). Both nucleotide binding domains were shown to bind ATP (1), and both are essential for function as mutation of either eliminated drug transport from cells (4) and ATPase activity (32), suggesting that the two sites were coupled allosterically. Hydrolysis of ATP was shown not to involve transient phosphorylation of the protein, and measurable catalysis was inhibited by orthovanadate [Vi(57)], which occupies the position of the liberated 
-phosphate in a ternary complex with P-glycoprotein and ADP, mimicking the transition state intermediate. These initial data were interpreted as evidence for a two-step mechanism with each catalytic event coupled to distinct stages (51). However, structural data has necessitated reinterpretation of the biochemistry data, resulting in a fundamental change to the perceived transport model. First, the structures of isolated NBDs were solved [the structure of HisP was reported in 1998 (18)], then dimers of NBDs in different physiological conformations, and finally of ABC transporters complete with NBDs and TMDs.
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Dimeric NBD Structures Hint at the Molecular Mechanism
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Purified NBDs isolated from their cognate TMDs are generally monomeric. Those that are homodimeric within the transport complex in vivo readily dimerise to hydrolyse ATP, but the association is transient (21, 41, 43, 64). Structures of NBDs in their catalytic conformation have therefore been difficult to achieve. However, structures have been determined for the NBD from the hemolysin exporter HlyB (65) and MJ0796 (52) in the presence of ATP, but only after mutation of the NBD to abrogate hydrolysis. These "closed NBD dimer" structures show the NBDs closed around two molecules of ATP at the interface (FIGURE 2A). The presence of two molecules of ATP is significant because it implies that the binding pockets act in concert and that the energy derived from ATP binding and the formation of the closed NBD dimer is used in a single step of the transport cycle. Similar structures have been observed with GlcV (60), MalK (7), and the nontransporter ABC NBDs Rad50 (15) and MutS (44); and with the conserved motifs of the NBDs involved in important protein-ATP or protein-protein contacts, it is clear that this is a physiological state (FIGURE 2B). There is also considerable evidence from studies with P-glycoprotein and CFTR to suggest that, in vivo, the TMDs of P-glycoprotein share an interface at the intracellular face of the membrane (34, 54) and that the NBDs interact directly (31, 40). The nucleotide binding pockets are therefore composite sites with both NBDs contributing to each site. In the dimeric state ATP is bound predominantly by the core subdomain, and the Walker A and B motifs, the stacking aromatic (as it has become known for its 
- interaction with the adenosine ring of the ATP), and the His-loop histidine all contact the bound nucleotide. The Q-loop glutamine also contacts the -phosphate of ATP via a water molecule. The Q-loop motif has been extended in Figure 2B where it is colored blue to illustrate that it originates from a helix on the top surface of the NBD where it interdigitates with the intracellular loops from the TMDs (see below). It also extends COOH terminally to connect the core subdomain of the NBD to the 
-helical subdomain. The -helical subdomain contains the ABC signature that contacts the second ATP (colored gray in FIGURE 2B), which is bound predominantly by the core subdomain of the associated blue NBD. Finally, the D-loop of the core subdomain may form a hydrogen bond with the back bone of the Walker A motif of the blue NBD.
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Conformational Change at the NBDs
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The position of the Q-loop is important because it is in a position to couple the ligand-binding sites within the TMDs to the ATP-binding sites of the NBDs. It also links the two subdomains of the NBD and therefore the two composite ATP-binding sites offering a molecular explanation for the observed allostery between the ATP catalytic sites (8, 28, 43, 64). The Q-loop has been suggested to move as ATP is bound consistent with a coupling role; however, this conclusion is drawn from comparison of monomeric nucleotide-free and dimeric ATP-bound NBDs (52, 65). Only in the case of MalK, the NBD of the maltose transporter from E. coli , are nucleotide-free NBDs dimeric in solution (7). This is because MalK has an additional regulatory domain that maintains an interface in the nucleotide-free form (FIGURE 3). Comparison of these two MalK structures suggests that the domains make a major conformational change as ATP is bound, likened to the action of tweezers. However, in common with the other structures of isolated NBDs, this interpretation does not take into account the influence of the TMDs on the extent of the conformational change at the NBDs. In BtuCD and HI1470/1, where each domain is on a separate polypeptide, the complex remains intact in the absence of ATP and, given that isolated NBDs only interact transiently during ATP hydrolysis, maintenance of the transport complex is mediated by NBD-TMD contacts.
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FIGURE 3. ATP-dependent conformational changes in an NBD dimer
The ATP-free (left) and ATP-bound (right) of MalK the NBD of the maltose importer from E.coli are shown [pdb files 1Q1E and 1Q12, respectively (7)]. The ATP-free conformation of MalK remains dimeric because the additional regulatory domains (colored pink and cyan) attached to their respective NBDs (colored gold and blue) maintains an interface in this state. In the ATP-bound form, the -helical subdomain containing the ABC signature (green spheres) is rotated forward with respect to the core subdomain of the same NBD to form a composite ATP-binding site with the Walker A motif of the second NBD. The Q-loop, which links the two subdomains and also makes contact with the -phosphate of the ATP is though to be instrumental in controlling this conformational change, but the extent of the conformational change is questionable because the TMDs, which would contact the top surface of the NBDs and likely constrain their separation, are absent.
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The precise nature of the open and closed NBD dimer conformations, therefore, awaits structure determination of the same full-size transporter in different physiological conformations in the presence and absence of ATP. However, circumstantial but persuasive corroboration that the Q-loop is the site of major conformational change and coupling between the energy-converting domains and the ligand-binding moiety of ABC proteins is found in the SMC proteins in which the Q-loop is the site of insertion of large-coiled coil domains, which extend from the NBDs to bind chromosomes (13).
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Structural Data from Full-Size Transporters
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At present, there are three complete structures in the database following the recent retraction of three distinct structures of MsbA (6). The domain arrangement for the remaining three full-length ABC transporters, is shown in Figure 4A. All are of bacterial origin; two are homologous PBP-dependent importers, BtuCD from E.coli , which transports vitamin B12 (30), and HI1470/1 from Haemophilus influenzae,which transports a metal-chelate (45), and the third is a drug exporter, Sav1866 from Staphlococcus aureus (9). The four core domains of the importers are on separate polypeptides and form a complex of homodimeric TMDs and homodimeric NBDs. Sav1866 is a "half transporter" with one NBD and one TMD per polypeptide, which functions as a homodimer. The homologous TMDs of the vitamin B12 and metal-chelate importers, BtuC and HI1471, respectively, and the non-homologous TMD of Sav1866, cross the membrane as -helices, but the similarity ends there. BtuC and HI1471 span the membrane 10 times per domain, whereas Sav1866 spans the membrane 6 times per domain. The intracellular loops (ICLs) of BtuC and HI1471 are short with the NBDs close to the inner leaflet of the membrane, whereas the ICLs of Sav1866 are much longer, extending 40 Å into the cytoplasm as helical extensions of the transmembrane helices.
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FIGURE 4. Domain organization of full-size ABC transporters
A: cartoon representations of the three full-size structures for ABC transporters; Sav1866 is crystallized with ADP (pdb 2HYD); BtuCD with tetravanadate (pdb 1L7V); and HI1470/1 in the absence of nucleotide (pdb 2NQ2). The TMDs are shown in dark blue and gray for the homologous BtuCD and HI1470/1, and red and teal for the non-homologous Sav1866. The NBDs, which are homologous throughout, are shown in gold and slate blue. Sav1866 is a "half transporter" and functions as a homodimer of a two-domain polypeptide (the red TMD is fused to the gold NBD). In BtuCD and HI1470/1, each domain is on a different polypeptide and the NBDs and TMDs are homodimers. The Walker A motif of the gold NBD and the ABC signature motif of the blue NBD are shown as spheres and colored red and green, respectively. These should be able to come together to coordinate nucleotide between them. B: ribbon model of Sav1866 rotated 90 degrees to the left with respect to that shown in A to illustrate the composite helical bundles in the TMDs. C: surface rendering of the NBD dimers of Sav1866, BtuCD, and HI1470/1 viewed from the membrane and showing the footprint (all residues within 4 Å) made by the ICLs of the red TMD for Sav1866 or blue TMD in the case of BtuCD and HI1470/1. For Sav1866, the footprint of ICL1 is shown in green and ICL2 in pink. The footprints made by the amino-terminal tail, IL3 and IL4 (the only ones within 4 Å of the surface) of BtuC are colored in cyan, yellow, and purple, respectively. His92 of the NBD is close to both the amino-terminal tail and IL3 and is coloured pale green. The footprints made by IL1 and IL3 and IL4 of HI1471 are colored in red, yellow, and purple, respectively (residues 142–145 of IL2 of HI1471 are not resolved; therefore, this analysis is likely to be incomplete). Overlap between the footprints of IL3 and IL4 is indicated in pink. Nucleotide and tetravanadate are shown in stick form and colored elementally throughout.
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The structures of BtuCD and HI1470/1 are similar but not identical. Essentially, they are both crystallized in the absence of nucleotide (BtuCD has tetra-vanadate in both nucleotide binding pockets, but the NBDs are not in the closed NBD conformation expected when ATP is bound). At the centre of the TMD-TMD interface, an aqueous chamber is evident, which is open at the extracellular face of the protein but tapers to close toward the intracellular face. The structure of BtuF (the PBP for BtuCD) in complex with vitamin B12 can be docked onto the BtuCD structure in a plausible, in silico experiment (5), suggesting that the conformation of the transport complex is in an outward configuration appropriate for binding of the BtuF-B12 complex from the periplasm. The TMDs of HI1470/1 also describe an aqueous chamber in what would be the plane of the membrane, but, in contrast to BtuCD, the chamber is open at the intracellular face of the protein, suggesting an inward-facing aspect for release of the ligand intracellularly. These two structures may therefore represent good models of the conformational changes expected in the TMDs during the transport process. However, the very subtle differences at the NBDs, with neither structure in the closed NBD conformation, means that hypotheses of how conformational changes in the TMDs are controlled by the ATP catalytic cycle, and vice versa, must remain speculative.
The domain organization of the importers is not shared with the multidrug resistance (MDR) class of exporter. The three structural models each have a plausible TMD-TMD interface and a NBD-NBD interface capable (or within a small conformational change) of coordinating nucleotide. Indeed, Sav1866 has ADP and BtuCD has tetra-vanadate mimicking nucleotide in an appropriate position. However, comparison of the interdomain architecture of the PBP-dependent importers with Sav1866 suggests that they may not be compatible. The TMD-TMD interface of Sav1866 is rotated with respect to the NBD-NBD interface, such that each TMD contacts both NBDs. This is readily illustrated by the footprints that the ICLs of one TMD make on the surface of the NBD dimer ( FIGURE 4C
). In Sav1866, ICL1 of the red TMD spans the composite ATP-binding site formed by the core subdomain of the gold NBD and the -helical subdomain of the slate blue NBD (the footprint is shown in green). ICL2 of the red TMD of Sav1866 makes contact exclusively with the slate blue NBD and not to the orange NBD to which it is fused in a single polypeptide. In the BtuCD and HI1470/1 structures, each TMD contacts only one NBD (illustrated in FIGURE 4C by the patchwork coloring of the surface of the NBD-NBD dimer of all residues within 4 Å of the ICLs of the blue TMD). Assessing the veracity of each of these distinct arrangements with the current information is difficult. There is no biochemical evidence that the TMDs of Sav1866 make contact with an NBD other than the one to which it is attached covalently. Also, when the different domains of the MDR class P-glycoprotein were expressed as individual proteins, NBD1 only interacted with TMD1, and NBD2 only interacted with TMD2 (33), suggesting that, if the TMDs interact with both NBDs during the transport cycle, it may only be transient and perhaps only within the complete, four-domain transport complex.
The helical bundles of the TMDs of the PBP-dependent importers and Sav1866 are also distinct. In BtuCD and HI1470/1, the 10 -helices of each TMD form a single bundle (FIGURE 4A), and although the two bundles share an interface in the membrane, they presumably fold and cross the membrane independently of each other. As illustrated by a different view of Sav1866 in Figure 4B, there are also two helical bundles in the TMDs of Sav1866, but these are composite bundles formed by the interaction of two TM helices from one TMD (helices 4 and 5 ) with four TM helices from the other (helices 1', 2',3', and 6'). Such a conformation for Sav1866, very different from the two examples of the PBP-dependent importers, will have to be tested to confirm its physiological veracity.
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Could the PBP-Importers and the Drug Exporters Have Distinct Transport Mechanisms?
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Sav1866 is crystallized with two molecules of ADP at the NBD-NBD interface. However, the NBD dimer interface of the Sav1866 structure closely resembles the ATP-bound conformations of bacterial NBDs crystallized in absence of their cognate TMDs (9). It is difficult to see how ADP can exit the Sav1866 structure unless there is further conformational change. Although this could, perhaps, be induced by binding of ligand, it may be better to consider Sav1866 as an ATP-bound model, but is it feasible that the protein moves between the Sav1866-like conformation (mimicking the ATP-bound state) to conformations more consistent with the two structures of the PBP-dependent importers? This would involve an extremely large change at the NBD-TMD interface, perhaps requiring uncoupling of the NBD from the TMD before reengagement. At much lower resolution but working with a full-size ABC transporter, evidence for large-scale conformational changes in the TMDs following ATP binding, and again after hydrolysis, has been obtained for P-glycoprotein (47). This evidence would seem to imply conformational change at the NBDs on the scale suggested by the MalK studies described above, but equally may be due to subtle changes in conformation at the NBDs transduced over a large distance (the intracellular loops of Sav1866, which is homologous with P-glycoprotein extend at least 40 Å from the lipid bilayer). In truth, there is insufficient data to describe, in detail with confidence, conformational changes that occur during the transport cycle, but at present it would be best to regard the MDR class of ABC exporters and the PBP-dependent importers as having different molecular mechanisms for ligand translocation through the membrane. The function of ABC transporters may be best thought of as the coupling of two distinct cycles: an ATP catalytic cycle at the NBDs and a ligand transport cycle at the TMDs. The conformational changes at the NBDs, switching between a nucleotide-free open conformation and an ATP-bound closed conformation, are likely to be conserved throughout the family. However, if the three available structures are physiologically relevant, the different classes of TMDs are likely to have evolved different mechanisms to couple the conformational changes of the NBD dimer to ligand translocation.
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Mechanism of Action of an MDR Exporter
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Extensive biochemical studies of the ATP catalytic cycle linked to pharmacological studies of drug affinity make P-glycoprotein arguably the best characterized ABC transporter. These data, now interpreted in light of the structural data described above, are the basis for the ATP-switch model to explain the mechanism of drug transport via P-glycoprotein (12). Essentially, transporters must cycle between high-and low-affinity states for ligand on different sides of the membrane. The ATP switch mechanism describes how these states are coupled to the ATP catalytic cycle in a way that is consistent with the structural data available (FIGURE 5).
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FIGURE 5. A simple ATP-switch mechanism powers ABC transporters
Ligand binding to a high-affinity pocket formed by the TMDs induces a conformational change in the NBDs resulting in a higher affinity for ATP. Two molecules of ATP bind to the NBDs. The energy released by the formation of the closed NBD dimer causes conformational change in the TMDs (which is sufficient for P-glycoprotein to extrude the anticancer drug vinblastine, although subsequent conformational changes may drive translocation of different drugs). ATP hydrolysis (an inexorable consequence of the closed conformation) triggers dissolution of the closed NBD dimer resulting in further conformational changes in the TMDs. Finally, phosphate and then ADP release restores the transporter to the open NBD dimer conformation ready for the subsequent cycle.
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Step I
Ligand binds to the TMDs in the high-affinity open NBD dimer conformation, inducing increased affinity for ATP.
In the nucleotide-free form, ABC transporters have a high affinity for ligand [this has been clearly demonstrated for drug transporters P-glycoprotein and LmrA (38, 49, 58)]. Binding of ligand to the TMDs causes a conformational change in the NBDs (24, 29, 35, 36, 42, 53), transduced via the ICLs of the TMDs, which interdigitate with the NBDs. The close proximity of the ICLs with the Q-loop and Walker A motifs suggests that the binding of ligand could directly influence the affinity of the NBDs for ATP, as suggested by structural studies of some crystals of HlyB and LolD with unusual conformations of the Walker A motif, which would preclude binding of ATP (59, 65), although as a mechanism this is by no means clear. Evidence for an increase in affinity for ATP in the presence of ligand has been difficult to measure and is largely indirect. For example, two-point mutations in TMDs of the maltose importer from E. coli uncouple ATP catalysis from the binding of its PBP-maltose complex and cause a 30-fold increase in affinity for ATP (36). An increase in affinity for ATP following ligand binding has also been measured for the DNA repair enzyme MutS (25) and for P-glycoprotein, albeit subtly (46).
Step II
ATP binding induces formation of the closed NBD dimer, which in turn induces a large conformational change in the TMDs sufficient to translocate ligand.
Structural data show two molecules of ATP at the NBD dimer interface, implying that they act in concert at a single step. Formation of the NBD dimer closed around the ATP has been calculated to generate a significant amount of free energy (52), and several distinct measurements suggest that this available energy is used to reconfigure the TMDs (24, 35, 36, 53). The extent of this conformational change is large and can be visualized at low resolution for P-glycoprotein (47). This may involve breaking interactions between TM -helices and formation of new contacts with different partners. Not surprisingly, this rearrangement can alter both the position and affinity of the ligand binding site. In P-glycoprotein, binding of nonhydrolysable analogs of ATP is sufficient to reduce the affinity for the drug vinblastine and so must effect transport of this drug at this stage (38). In CFTR, ATP binding in the absence of hydrolysis is sufficient to open the chloride channel (61), and in MutS conformational changes associated with ATP binding allows the repair complex to form (11, 25).
Step III
ATP hydrolysis initiates dissolution of the closed NBD dimer.
The transient nature of isolated NBD dimers induced by ATP suggests that hydrolysis is an inexorable consequence of closed NBD dimer formation. The mechanism of hydrolysis is contentious with base catalysis proposed for LolD (52) and substrate-assisted catalysis proposed for HlyD (63). Whichever mechanism, it is clear that hydrolysis destabilizes the closed NBD dimer. For some ABC transporters like P-glycoprotein, hydrolysis of both ATPs is necessary for completion of the transport cycle (4), and the ATPs are hydrolysed nonsimultaneously (16, 17, 51, 56). The two ATP binding pockets of P-glycoprotein have been shown to be biochemically asymmetric, but it is not yet clear whether this is a cause of nonsimultaneous hydrolysis or the result of it (56). In other ABC transporters, such as in MRP1 and CFTR, hydrolysis of only one ATP may be sufficient to drive the protein through the conformational cycle (3, 62). The post-hydrolytic state can be shunted into a stable conformation once phosphate has left the complex, as it can be replaced by vanadate. This form of the protein, which may mimic the ADP.Pi state, has a conformation distinct from the ATP-bound and nucleotide-free forms (34, 47) and may be coupled to transport from different drug binding pockets in Pgp (26, 49), although for vinblastine the protein remains in the low-affinity state (37).
Step IV
Pi then ADP is released to complete the transport cycle and restore the protein to a high-affinity state for ligand.
Following hydrolysis, the liberated phosphate is released from the protein. Again two mechanisms have been postulated for this based on the analysis of the structures of HlyB [via exit channels (65)] and LolD [following electrostatic repulsion of the two NBDs (52)]. The quick release of phosphate, which allows the protein to be trapped with ADP and vanadate, correlates well with the restoration of high-affinity vinblastine binding to P-glycoprotein, indicating that the conformational change associated with phosphate release is also coupled to the ligand binding sites (38). Affinity of ABC tranporters for ADP is low, and ADP is unable to stabilize the dimeric interaction of isolated NBDs (which is one reason for doubting the absolute veracity of the ADP bound structure of Sav1866); however, at least for MutS, it appears that ADP remains bound until DNA binding induces an exchange for ATP (11).
The ATP-switch model is the product of biochemical data interpreted in light of recent advances in structure determination for several ABC transporters. The model is divided into four steps, or four conformational changes; the first associated with the binding of ligand, then three that make use of the free energy available from protein-ATP and protein-protein interactions associated with ATP binding, ATP hydrolysis, and ADP.Pi release. Any of the latter three steps could be used to transport ligand depending on the nature of the ligand binding site, so P-glycoprotein could potentially translocate different drugs at different stages of the cycle. It is also important to appreciate that each step is closely coupled to the next; for example, formation of the closed NBD dimer is likely to be inexorably linked to ATP hydrolysis, just as the transport of vinblastine by P-glycoprotein appears to be.
Although some important details remain to be elucidated (for example, the precise nature of the ligand binding sites), this general mechanism of the ATP switch allows minor modifications to explain the idiosyncratic behavior of different ABC transporters with different classes of TMD or ABC NBDs coupled to functions other than transport.
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References
|
|---|
- al-ShawiMK, Urbatsch IL, Senior AE. Covalent inhibitors of P-glycoprotein ATPase activity. J Biol Chem 269: 8986–8992, 1994.[Abstract/Free Full Text]
- AlbrechtC, McVey JH, Elliott JI, Sardini A, Kasza I, Mumford AD, Naoumova RP, Tuddenham EG, Szabo K, Higgins CF. A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome. Blood 106: 542–549, 2005.[Abstract/Free Full Text]
- Aleksandrov AA, Chang X, Aleksandrov L, Riordan JR. The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating. J Physiol 528: 259–265, 2000.[Abstract/Free Full Text]
- AzzariaM, Schurr E, Gros P. Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance. Mol Cell Biol 9: 5289–5297, 1989.[Abstract/Free Full Text]
- BorthsEL, Locher KP, Lee AT, Rees DC. The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter. Proc Natl Acad Sci USA 99: 16642–16647, 2002.[Abstract/Free Full Text]
- ChangG, Roth CB, Reyes CL, Pornillos O, Chen YJ, Chen AP. Retraction. Science 314: 1875, 2006.[ISI][Medline]
- ChenJ, Lu G, Lin J, Davidson AL, Quiocho FA. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol Cell 12: 651–661, 2003.[CrossRef][ISI][Medline]
- DavidsonAL, Laghaeian SS, Mannering DE. The maltose transport system of Escherichia coli displays positive cooperativity in ATP hydrolysis. J Biol Chem 271: 4858–4863, 1996.[Abstract/Free Full Text]
- DawsonRJ, Locher KP. Structure of a bacterial multidrug ABC transporter. Nature 443: 180–185, 2006.[CrossRef][Medline]
- DeanM, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11: 1156–1166, 2001.[Abstract/Free Full Text]
- GradiaS, Subramanian D, Wilson T, Acharya S, Makhov A, Griffith J, Fishel R. hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA. Mol Cell 3: 255–261, 1999.[CrossRef][ISI][Medline]
- HigginsCF, Linton KJ. The ATP switch model for ABC transporters. Nat Struct Mol Biol 11: 918–926, 2004.[CrossRef][ISI][Medline]
- HiranoT. At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7: 311–322, 2006.[CrossRef][ISI][Medline]
- HollandIB, Cole SPC, Kuchler K, Higgins CF, ABC. Proteins: From Bacteria to Man. London: Academic Press, 2003.
- HopfnerKP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, Tainer JA. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101: 789–800, 2000.[CrossRef][ISI][Medline]
- HrycynaCA, Ramachandra M, Ambudkar SV, Ko YH, Pedersen PL, Pastan I, Gottesman MM. Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites. J Biol Chem 273: 16631–16634, 1998.[Abstract/Free Full Text]
- HrycynaCA, Ramachandra M, Germann UA, Cheng PW, Pastan I, Gottesman MM. Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry 38: 13887–13899, 1999.[CrossRef][Medline]
- HungLW, Wang IX, Nikaido K, Liu PQ, Ames GF, Kim SH. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396: 703–707, 1998.[CrossRef][Medline]
- InagakiN, Gonoi T, Clement JPt Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270: 1166–1170, 1995.[Abstract/Free Full Text]
- Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. Clin Liver Dis 4: 753–763, 2000.[CrossRef][Medline]
- JanasE, Hofacker M, Chen M, Gompf S, van der Does C, Tampe R. The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p. J Biol Chem 278: 26862–26869, 2003.[Abstract/Free Full Text]
- JonkerJW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH, Schinkel AH. Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan. J Natl Cancer Inst 92: 1651–1656, 2000.[Abstract/Free Full Text]
- KozakL, Gopal G, Yoon JH, Sauna ZE, Ambudkar SV, Thakurta AG, Dhar R. Elf1p, a member of the ABC class of ATPases, functions as a mRNA export factor in Schizosacchromyces pombe. J Biol Chem 277: 33580–33589, 2002.[Abstract/Free Full Text]
- KreimerDI, Chai KP, Ferro-Luzzi Ames G. Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex. Biochemistry 39: 14183–14195, 2000.[CrossRef][Medline]
- LamersMH, Winterwerp HH, Sixma TK. The alternating ATPase domains of MutS control DNA mismatch repair. EMBO J 22: 746–756, 2003.[CrossRef][ISI][Medline]
- LintonKJ, Higgins CF. Structure and function of ABC transporters: the ATP switch provides flexible control. Pflügers Arch 453: 555–567, 2007.[CrossRef][ISI][Medline]
- LintonKJ, Higgins CF. The Escherichia coli ATP-binding cassette (ABC) proteins. Mol Microbiol 28: 5–13, 1998.[CrossRef][ISI][Medline]
- LiuCE, Liu PQ, Ames GF. Characterization of the adenosine triphosphatase activity of the periplasmic histidine permease, a traffic ATPase (ABC transporter). J Biol Chem 272: 21883–21891, 1997.[Abstract/Free Full Text]
- LiuR, Sharom FJ. Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. Biochemistry 35: 11865–11873, 1996.[CrossRef][Medline]
- LocherKP, Lee AT, Rees The E DC. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296: 1091–1098, 2002.[Abstract/Free Full Text]
- LooTW, Bartlett MC, Clarke DM. The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. J Biol Chem 277: 41303–41306, 2002.[Abstract/Free Full Text]
- LooTW, Clarke DM. Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity. J Biol Chem 270: 22957–22961, 1995.[Abstract/Free Full Text]
- LooTW, Clarke DM. P-glycoprotein. Associations between domains and between domains and molecular chaperones. J Biol Chem 270: 21839–21844, 1995.[Abstract/Free Full Text]
- LooTW, Clarke DM. The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis. J Biol Chem 275: 5253–5256, 2000.[Abstract/Free Full Text]
- ManciuL, Chang XB, Buyse F, Hou YX, Gustot A, Riordan JR, Ruysschaert JM. Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione. J Biol Chem 278: 3347–3356, 2003.[Abstract/Free Full Text]
- ManneringDE, Sharma S, Davidson AL. Demonstration of conformational changes associated with activation of the maltose transport complex. J Biol Chem 276: 12362–12368, 2001.[Abstract/Free Full Text]
- MartinC, Berridge G, Mistry P, Higgins C, Charlton P, Callaghan R. Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis. Biochemistry 39: 11901–11906, 2000.[CrossRef][Medline]
- MartinC, Higgins CF, Callaghan R. The vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein. Biochemistry 40: 15733–15742, 2001.[CrossRef][Medline]
- Martinez-MirA, Paloma E, Allikmets R, Ayuso C, del Rio T, Dean M, Vilageliu L, Gonzalez-Duarte R, Balcells S. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR. Nat Genet 18: 11–12, 1998.[CrossRef][ISI][Medline]
- MenseM, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J 25: 4728–4739, 2006.[CrossRef][ISI][Medline]
- MoodyJE, Millen L, Binns D, Hunt JF, Thomas PJ. Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters. J Biol Chem 277: 21111–21114, 2002.[Abstract/Free Full Text]
- NeumannL, Abele R, Tampe R. Thermodynamics of peptide binding to the transporter associated with antigen processing (TAP). J Mol Biol 324: 965–973, 2002.[CrossRef][ISI][Medline]
- NikaidoK, Liu PQ, Ames GF. Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity. J Biol Chem 272: 27745–27752, 1997.[Abstract/Free Full Text]
- ObmolovaG, Ban C, Hsieh P, Yang W. Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407: 703–710, 2000.[CrossRef][Medline]
- PinkettHW, Lee AT, Lum P, Locher KP, Rees DC. An inward-facing conformation of a putative metal-chelate-type ABC transporter. Science 315: 373–377, 2006.[CrossRef][ISI][Medline]
- QuQ, Russell PL, Sharom FJ. Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle. Biochemistry 42: 1170–1177, 2003.[CrossRef][Medline]
- RosenbergMF, Velarde G, Ford RC, Martin C, Berridge G, Kerr ID, Callaghan R, Schmidlin A, Wooding C, Linton KJ, Higgins CF. Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle. EMBO J 20: 5615–5625, 2001.[CrossRef][ISI][Medline]
- SarkadiB, Homolya L, Szakacs G, Varadi A. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system. Physiol Rev 86: 1179–1236, 2006.[Abstract/Free Full Text]
- SaunaZE, Ambudkar SV. Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein. Proc Natl Acad Sci USA 97: 2515–2520, 2000.[Abstract/Free Full Text]
- SchinkelAH, Mol CA, Wagenaar E, van Deemter L, Smit JJ, Borst P. Multidrug resistance and the role of P-glycoprotein knockout mice. Eur J Cancer 31A: 1295–1298, 1995.[CrossRef]
- SeniorAE, al-Shawi MK, Urbatsch IL. The catalytic cycle of P-glycoprotein. FEBS Lett 377: 285–289, 1995.[CrossRef][ISI][Medline]
- SmithPC, Karpowich N, Millen L, Moody JE, Rosen J, Thomas PJ, Hunt JF. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol Cell 10: 139–149, 2002.[CrossRef][ISI][Medline]
- SonveauxN, Vigano C, Shapiro AB, Ling V, Ruysschaert JM. Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis. J Biol Chem 274: 17649–17654, 1999.[Abstract/Free Full Text]
- StenhamDR, Campbell JD, Sansom MS, Higgins CF, Kerr ID, Linton KJ. An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling. FASEB J 17: 2287–2289, 2003.[Abstract/Free Full Text]
- TabcharaniJA, Chang XB, Riordan JR, Hanrahan JW. Phosphorylation-regulated Cl– channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352: 628–631, 1991.[CrossRef][Medline]
- TomblineG, Muharemagic A, White LB, Senior AE. Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway. Biochemistry 44: 12879–12886, 2005.[CrossRef][Medline]
- UrbatschIL, Sankaran B, Weber J, Senior AE. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J Biol Chem 270: 19383–19390, 1995.[Abstract/Free Full Text]
- van VeenHW, Margolles A, Muller M, Higgins CF, Konings WN. The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism. EMBO J 19: 2503–2514, 2000.[CrossRef][ISI][Medline]
- VerdonG, Albers SV, Dijkstra BW, Driessen AJ, Thunnissen AM. Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations. J Mol Biol 330: 343–358, 2003.[CrossRef][ISI][Medline]
- VerdonG, Albers SV, van Oosterwijk N, Dijkstra BW, Driessen AJ, Thunnissen AM. Formation of the productive ATP-Mg2+-bound dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus. J Mol Biol 334: 255–267, 2003.[CrossRef][ISI][Medline]
- VerganiP, Lockless SW, Nairn AC, Gadsby DC. CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433: 876–880, 2005.[CrossRef][Medline]
- YangR, Cui L, Hou YX, Riordan JR, Chang XB. ATP binding to the first nucleotide binding domain of multidrug resistance-associated protein plays a regulatory role at low nucleotide concentration, whereas ATP hydrolysis at the second plays a dominant role in ATP-dependent leukotriene C4 transport. J Biol Chem 278: 30764–30771, 2003.[Abstract/Free Full Text]
- ZaitsevaJ, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J 24: 1901–1910, 2005.[CrossRef][ISI][Medline]
- ZaitsevaJ, Jenewein S, Wiedenmann A, Benabdelhak H, Holland IB, Schmitt L. Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter. Biochemistry 44: 9680–9690, 2005.[CrossRef][Medline]
- ZaitsevaJ, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, Schmitt L. A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. EMBO J 25: 3432–3443, 2006.[CrossRef][ISI][Medline]
- ZhouS, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7: 1028–1034, 2001.[CrossRef][ISI][Medline]
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