Powering the peptide pump: TAP crosstalk with energetic nucleotides

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with āntigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.     

Functional non-equivalence of ATP-binding cassette signature motifs in the transporter associated with antigen processing (TAP)

The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.     

Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.     

Functional asymmetry of the ATP-binding-cassettes of the ABC transporter TAP is determined by intrinsic properties of the nucleotide binding domains.

The ATP-binding-cassette (ABC) transporter associated with antigen processing (TAP) delivers peptides into the ER. TAP consists of two polypeptides (TAP1 and TAP2) each with an N-terminal transmembrane (TMD) and a C-terminal nucleotide binding domain (NBD). The two highly homologous NBDs of TAP show different nucleotide binding specificites, and identical mutations in the domains can have different effects on peptide transport. We asked whether this functional asymmetry of the NBDs is an intrinsic property or is imposed by the TMDs to which they are linked. To investigate the functional interdependence of the TAP domains, we created various TAP variants in which TMDs and/or NBDs were exchanged. All TAP variants except those with two TMDs of TAP1 could assemble. The TMDs did not affect the different nucleotide binding properties of the NBDs. The TAP variant with switched NBDs showed active peptide transport while the variants with pairs of identical NBDs or TMDs were inactive. Although both types of TMDs and NBDs have to be present for peptide transport they do not have to be assorted as in wild-type TAP. Thus, TAP domains seem to preserve functional autonomy despite their fusion into single polypeptide chains. We propose that the two NBDs act as nonequivalent 'modules' that directly determine the functional asymmetry of the included ATP-binding-cassettes. This provides a new insight into the function of NBDs and opens up new possibilities to investigate the molecular mechanism of the 'NBD engine' in ABC transporters.     

Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.     

Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.     

The distinct nucleotide binding states of the transporter associated with antigen processing (TAP) are regulated by the nonhomologous C-terminal tails of TAP1 and TAP2.

The transporter associated with antigen processing (TAP) delivers peptides into the lumen of the endoplasmic reticulum for binding onto major histocompatibility complex class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each with an N-terminal transmembrane domain and a C-terminal cytosolic nucleotide binding domain (NBD). The two NBDs have distinct intrinsic nucleotide binding properties. In the resting state of TAP, the NBD1 has a much higher binding activity for ATP than the NBD2, while the binding of ADP to the two NBDs is equivalent. To attribute the different nucleotide binding behaviour of NBD1 and NBD2 to specific sequences, we generated chimeric TAP1 and TAP2 polypeptides in which either the nonhomologous C-terminal tails downstream of the Walker B motif, or the core NBDs which are enclosed by the conserved Walker A and B motifs, were reciprocally exchanged. Our biochemical and functional studies on the different TAP chimeras show that the distinct nucleotide binding behaviour of TAP1 and TAP2 is controlled by the nonhomologous C-terminal tails of the two TAP chains. In addition, our data suggest that the C-terminal tail of TAP2 is required for a functional transporter by regulating ATP binding. Further experiments indicate that ATP binding to NBD2 is important because it prevents simultaneous uptake of ATP by TAP1. We propose that the C-terminal tails of TAP1 and TAP2 play a crucial regulatory role in the coordination of nucleotide binding and ATP hydrolysis by TAP.     

Adventures with ABC-proteins: highly conserved ATP-dependent transporters

The general properties of ABC transporters, from bacteria to humans, including a brief history of their initial discovery, are considered. ABC transporters, one of the largest protein super families and vital for human health, are in toto responsible for the transport of an enormous range of molecules from ions (CFTR) or anti-tumour drugs (Pgp/MDR) to large polypeptides. Nevertheless, all ABC transporters are powered by a conserved ATPase the ABC or NBD domain, using in all probability the same basic mechanism of action for the hydrolysis of ATP and its coupling to the transport process. Based on recent high resolution structures of several NBDs and an intact transporter, a model of how dimers of these important proteins function will be discussed, with particular attention to HlyB, the ABC transporter from E. coli.     

ATP hydrolysis at one of the two sites in ABC transporters initiates transport related conformational transitions

ABC transporters play important roles in all types of organisms by participating in physiological and pathological processes. In order to modulate the function of ABC transporters, detailed knowledge regarding their structure and dynamics is necessary. Available structures of ABC proteins indicate three major conformations, a nucleotide-bound "bottom-closed" state with the two nucleotide binding domains (NBDs) tightly closed, and two nucleotide-free conformations, the "bottom-closed" and the "bottom-open", which differ in the extent of separation of the NBDs. However, it remains a question how the widely open conformation should be interpreted, and whether hydrolysis at one of the sites can drive conformational transitions while the NBDs remain in contact. To extend our knowledge, we have investigated the dynamic properties of the Sav1866 transporter using molecular dynamics (MD) simulations. We demonstrate that the replacement of one ATP by ADP alters the correlated motion patterns of the NBDs and the transmembrane domains (TMD). The results suggest that the hydrolysis of a single nucleotide could lead to extracellular closure, driving the transport cycle. Essential dynamics analysis of simulations suggests that single nucleotide hydrolysis can drive the system toward a "bottom-closed" apo conformation similar to that observed in the structure of the MsbA transporter. We also found significant structural instability of the "bottom-open" form of the transporters in simulations. Our results suggest that ATP hydrolysis at one of the sites promotes transport related conformational changes leading to the "bottom-closed" apo conformation, which could thus be physiologically more relevant for describing the structure of the apo state.     

The Human Transporter Associated with Antigen Processing: MOLECULAR MODELS TO DESCRIBE PEPTIDE BINDING COMPETENT STATES

The human transporter associated with antigen processing (TAP) is a member of the ATP binding cassette (ABC) transporter superfamily. TAP plays an essential role in the antigen presentation pathway by translocating cytosolic peptides derived from proteasomal degradation into the endoplasmic reticulum lumen. Here, the peptides are loaded into major histocompatibility class I molecules to be in turn exposed at the cell surface for recognition by T-cells. TAP is a heterodimer formed by the association of two half-transporters, TAP1 and TAP2, with a typical ABC transporter core that consists of two nucleotide binding domains and two transmembrane domains. Despite the availability of biological data, a full understanding of the mechanism of action of TAP is limited by the absence of experimental structures of the full-length transporter. Here, we present homology models of TAP built on the crystal structures of P-glycoprotein, ABCB10, and Sav1866. The models represent the transporter in inward- and outward-facing conformations that could represent initial and final states of the transport cycle, respectively. We described conserved regions in the endoplasmic reticulum-facing loops with a role in the opening and closing of the cavity. We also identified conserved π-stacking interactions in the cytosolic part of the transmembrane domains that could explain the experimental data available for TAP1-Phe-265. Electrostatic potential calculations gave structural insights into the role of residues involved in peptide binding, such as TAP1-Val-288, TAP2-Cys-213, TAP2-Met-218. Moreover, these calculations identified additional residues potentially involved in peptide binding, in turn verified with replica exchange simulations performed on a peptide bound to the inward-facing models.     

Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.     

Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB

ABC transporters are a large and important family of membrane proteins involved in substrate transport across the membrane. The transported substrates are quite diverse, ranging from monatomic ions to large biomolecules. Consequently, some ABC transporters are involved in biomedically relevant situations, from genetic diseases to multidrug resistance. The most conserved domains in ABC transporters are the nucleotide binding domains (NBDs), which form a dimer responsible for the binding and hydrolysis of ATP, concomitantly with substrate translocation. To elucidate how ATP hydrolysis structurally affects the NBD dimer, and consequently the transporter, we performed a molecular dynamics study on the NBD dimer of the HlyB ABC exporter. We have observed a change in the contact surface between the monomers after hydrolysis, even though we have not seen dimer opening in any of the five 100 ns simulations. We have also identified specific regions that respond to ATP hydrolysis, in particular the X-loop motif of ABC exporters, which has been shown to be in contact with the coupling helices of the transmembrane domains (TMDs). We propose that this motif is an important part of the NBD-TMD communication in ABC exporters. Through nonequilibrium analysis, we have also identified gradual conformational changes within a short time scale after ATP hydrolysis.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.     

The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p

The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a Km of 0.6 mm and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1.7), indicating that the functional state is a dimer. Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD. The nucleotide composition of trapped intermediate states was determined using [alpha-32P]ATP and [gamma-32P]ATP. Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode. Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides.     

The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact,Head-to-Tail Configuration of the Nucleotide-Binding Domains

ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration ‘sandwich’ dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD ‘Switch’ mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

Structure of ABC transporters

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.     

Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA

MsbA is a member of the ABC transporter superfamily and is homologous to ABC transporters linked to multidrug resistance. The nucleotide binding domains (NBDs) of these proteins include conserved motifs that are involved in ATP binding, including conserved SALD residues (D-loop) that are diagnostic in identifying ABC transporters but whose roles have not been identified. Within the D-loop, single point mutations L511P and D512G were discovered by random mutational analysis of MsbA to disrupt protein function in the cell [Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233] but have not been further studied in MsbA or in detail in any other ABC transporter. In these studies, we show that both L511P and D512G mutants of MsbA are able to bind ATP at near-wild-type levels but are unable to maintain cell viability in an in vivo growth assay, verifying the theory that they are dysfunctional at some point after ATP binding. An ATPase assay further suggests that the L511P mutation prevents effective ATP hydrolysis, and an ATP detection assay reveals that only small amounts of ATP are hydrolyzed; D512G is able to hydrolyze ATP at a rate 3-fold faster than that of the wild type. EPR spectroscopy studies using reporter sites within the NBDs also indicate that at least some hydrolysis occurs in L511P or D512G MsbA but show fewer spectral changes than observed for the same reporters in the wild-type background. These studies indicate that L511 is necessary for efficient ATP hydrolysis and D512 is essential for conformational rearrangements required for flipping lipid A.