Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.     

Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines KATP channel SUR2A catalytic activity

Nucleotide binding domains (NBDs) secure ATP-binding cassette (ABC) transporter function. Distinct from traditional ABC transporters, ABCC9-encoded sulfonylurea receptors (SUR2A) form, with Kir6.2 potassium channels, ATP-sensitive K+ (K ATP) channel complexes. SUR2A contains ATPase activity harbored within NBD2 and, to a lesser degree, NBD1, with catalytically driven conformations exerting determinate linkage on the Kir6.2 channel pore. While homodomain interactions typify NBDs of conventional ABC transporters, heterodomain NBD interactions and their functional consequence have not been resolved for the atypical SUR2A protein. Here, nanoscale protein topography mapped assembly of monodisperse purified recombinant SUR2A NBD1/NBD2 domains, precharacterized by dynamic light scattering. Heterodomain interaction produced conformational rearrangements inferred by secondary structural change in circular dichroism, and validated by atomic force and transmission electron microscopy. Physical engagement of NBD1 with NBD2 translated into enhanced intrinsic ATPase activity. Molecular modeling delineated a complemental asymmetry of NBD1/NBD2 ATP-binding sites. Mutation in the predicted catalytic base residue, D834E of NBD1, altered NBD1 ATPase activity disrupting potentiation of catalytic behavior in the NBD1/NBD2 interactome. Thus, NBD1/NBD2 assembly, resolved by a panel of proteomic approaches, provides a molecular substrate that determines the optimal catalytic activity in SUR2A, establishing a paradigm for the structure-function relationship within the K ATP channel complex.     

Structure of the human multidrug resistance protein 1 nucleotide binding domain 1 bound to Mg2+/ATP reveals a non-productive catalytic site

Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.     

Structure-function relationships in an anion-translocating ATPase

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.     

Identification of the Distance between the Homologous Halves of P-glycoprotein That Triggers the High/Low ATPase Activity Switch

P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 Å). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 Å (α-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 Å, although cross-linking at distances greater than 20 Å yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 Å, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125–5141).     

The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.     

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.     

ABC transporter architecture and regulatory roles of accessory domains

We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties.     

Investigating the role of the invariant carboxylate residues E552 and E1197 in the catalytic activity of Abcb1a (mouse Mdr3)

The invariant carboxylate residue which follows the Walker B motif (hyd(4)DE/D) in the nucleotide-binding domains (NBDs) of ATP-binding cassette transporters is thought to be involved in the hydrolysis of the gamma-phosphate of MgATP, either by activating the attacking water molecule or by promoting substrate-assisted catalysis. In Abcb1a, this invariant carboxylate residue corresponds to E552 in NBD1 and E1197 in NBD2. To further characterize the role of these residues in catalysis, we created in Abcb1a the single-site mutants E552D, N and A in NBD1, and E1197D, N and A in NBD2, as well as the double-mutant E552Q/E1197Q. In addition, we created mutants in which the Walker A K --> R mutation known to abolish ATPase activity was introduced in the non-mutant NBD of E552Q and E1197Q. ATPase activity, binding affinity and trapping properties were tested for each Abcb1a variant. The results suggest that the length of the invariant carboxylate residue is important for the catalytic activity, whereas the charge of the side chain is critical for full turnover to occur. Moreover, in the double-mutants where the K --> R mutation is introduced in the 'wild-type' NBD of the E --> Q mutants, single-site turnover is observed, especially when NBD2 can undergo gamma-P(i) cleavage. The results further support the idea that the NBDs are not symmetric and suggest that the invariant carboxylates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.     

Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.     

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.     

Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.     

Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1) function by 5'-fluorosulfonylbenzoyl 5'-adenosine: evidence for an ATP analogue that interacts with both drug-substrate-and nucleotide-binding sites

5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.     

Expression and activity of the nucleotide-binding domains of the human ABCA1 transporter

The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.     

Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1

Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics. The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[(32)P]ATP and to trap 8-azido-[(32)P]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido-[(32)P]ATP, while trapping of 8-azido-[(32)P]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-[(32)P]ATP.     

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.     

Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.     

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.     

The ATPase Activity of the P-glycoprotein Drug Pump Is Highly Activated When the N-terminal and Central Regions of the Nucleotide-binding Domains Are Linked Closely Together

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 Å) or long (22 Å) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.