Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders

ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.     

Engineering ATPase activity in the isolated ABC cassette of human TAP1

The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.     

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.     

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.     

Both ATP sites of human P-glycoprotein are essential but not symmetric.

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.     

Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.     

ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.     

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.     

ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.     

Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.     

Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.     

Mg2+ -dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2)

ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419-15425]. Subsequent to the initial binding, Mg(2+) drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497-500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [(32)P]N(3)ATP (8-azido-ATP) and [(32)P]N(3)ADP (8-azido-ADP). Only N(3)ATP, but not N(3)ADP, can be bound initially at NBD1 in the absence of Mg(2+). Despite the lack of a requirement for Mg(2+) for ATP binding, retention of the NTP at 37 degrees C was dependent on the cation. However, at reduced temperature (4 degrees C), N(3)ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg(2+). Occlusion occurred identically in a DeltaNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.     

Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508

Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.     

Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1)

MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.     

ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.     

Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.     

Kinetics of the ATP hydrolysis cycle of the nucleotide-binding domain of Mdl1 studied by a novel site-specific labeling technique

We have recently proposed a "processive clamp" model for the ATP hydrolysis cycle of the nucleotide-binding domain (NBD) of the mitochondrial ABC transporter Mdl1 (Janas, E., Hofacker, M., Chen, M., Gompf, S., van der Does, C., and Tampé, R. (2003) J. Biol. Chem. 278, 26862-26869). In this model, ATP binding to two monomeric NBDs leads to formation of an NBD dimer that, after hydrolysis of both ATPs, dissociates and releases ADP. Here, we set out to follow the association and dissociation of NBDs using a novel minimally invasive site-specific labeling technique, which provides stable and stoichiometric attachment of fluorophores. The association and dissociation kinetics of the E599Q-NBD dimer upon addition and removal of ATP were determined by fluorescence self-quenching. Remarkably, the rate of ATP hydrolysis of the wild type NBD is determined by the rate of NBD dimerization. In the E599QNBD, however, in which the ATP hydrolysis is 250-fold reduced, the ATP hydrolysis reaction controls dimer dissociation and the overall ATPase cycle. These data explain contradicting observations on the rate-limiting step of various ABC proteins and further demonstrate that dimer formation is an important step in the ATP hydrolysis cycle.     

Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport

ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.     

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.