The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein

P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.     

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.     

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.     

Mutagenesis of the putative nucleotide-binding domains of the multidrug resistance associated protein (MRP). Analysis of the effect of these mutations on MRP mediated drug resistance and transport

Studies were conducted to examine the functional role of the nucleotide-binding domains of MRP in drug resistance and drug transport in isolated membrane vesicles. In vivo studies were conducted by preparing stable transfectants of HeLa cells with wild-type MRP cDNA or MRP cDNAs which had been mutated at certain nucleotide binding domains (NBD). Stable transfectants producing equivalent amounts of the MRP encoded protein P190 were used in this study. The results demonstrated that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to chemotherapeutic agents. Certain single-site mutations in lysines in these same motifs also reduce IC₅₀ values. It has also been observed that mutation of the MRP NBDs results in an increase in drug accumulation and a reduction in drug efflux. Additional studies have been carried out in which recombinant baculovirus containing either wild-type MRP or MRP containing mutated NBDs was prepared and used to infect SF21 insect cells. Using this system we have analyzed the effects of these mutations on in vitro transport of leukotriene C₄ (LTC₄) 17 β-estradiol 17 (β-D-glucuronide)(E₂17βG) and daunomycin in membrane vesicles prepared from baculovirus infected cells. The results demonstrate that deletions and site-specific mutations in MRP NBDs greatly reduce the ATP dependent transport of all three substrates. The results of these studies conducted both in vivo and in vitro demonstrate that the NBDs of MRP function in a cooperative manner and are critical for the transport activity of the MRP encoded protein P190. These studies also identify specific lysines in NBD1 and NBD2 which are important for optimal MRP activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein

Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [(3)H]leukotriene C(4) (LTC(4)), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH(2)-proximal site is preferentially inhibited by S-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14-17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the γ-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.     

Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport

Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.     

ATP-binding properties of P glycoprotein from multidrug-resistant KB cells

The photoaffinity reagent 8-azido-alpha-[32P]ATP was used to label a protein of 170 kDa in membrane vesicle preparations from a highly multidrug-resistant cell line, KB-V1, but not from the drug-sensitive parental cell line KB-3-1. The 170-kDa labeled protein was immunoprecipitated with a monoclonal antibody (MRK-16) to P glycoprotein. Both ATP and GTP inhibited labeling by 8-azido-alpha-[32P]ATP. Labeling of P170 was not inhibited by 5 mM ADP, 5 mM ribose-5-phosphate, or 100 microM vinblastine. These data directly demonstrate that P glycoprotein has a nucleotide-binding site that could supply energy for drug transport.     

Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1

Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.     

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 binding,not hydrolysis,at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.     

S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1).

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.     

Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.     

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.     

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.     

REV3 and REV1 play major roles in recombination-independent repair of DNA interstrand cross-links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA)

DNA interstrand cross-links (ICLs) are the most cytotoxic lesions to eukaryotic genome and are repaired by both homologous recombination-dependent and -independent mechanisms. To better understand the role of lesion bypass polymerases in ICL repair, we investigated recombination-independent repair of ICLs in REV3 and REV1 deletion mutants constructed in avian DT40 cells and mouse embryonic fibroblast cells. Our results showed that Rev3 plays a major role in recombination-independent ICL repair, which may account for the extreme sensitivity of REV3 mutants to cross-linking agents. This result raised the possibility that the NER gap synthesis, when encountering an adducted base present in the ICL repair intermediate, can lead to recruitment of Rev3, analogous to the recruitment of polymerase eta during replicative synthesis. Indeed, the monoubiquitination-defective Proliferating Cell Nuclear Antigen (PCNA) mutant exhibits impaired recombination-independent ICL repair as well as drastically reduced mutation rate, indicating that the PCNA switch is utilized to enable lesion bypass during DNA repair synthesis. Analyses of a REV1 deletion mutant also revealed a significant reduction in recombination-independent ICL repair, suggesting that Rev1 cooperates with Rev3 in recombination-independent ICL repair. Moreover, deletion of REV3 or REV1 significantly altered the spectrum of mutations resulting from ICL repair, further confirming their involvement in mutagenic repair of ICLs.     

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.     

Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis

MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.