ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.     

G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects

Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.     

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.     

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.     

囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

囊性纤维化跨膜电导调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是一种Cl^-通道,属于ATP结合（ATP-binding cassette,ABC）转运体超家族。CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化（cystic fibrosis,CF）发生的主要原因。这种疾病患者各组织上皮细胞内Cl^-转运失调。目前,与CF相关的不同突变超过1400种。CFTR调节（regulatory,R）域负责调控,核苷酸结合域（nucleotide-binding domains,NBDs）NBD1和NBD2负责ATP结合和水解门控。近期研究发现CFFR的NBDs与其它ABC蛋白一样可以二聚化。二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列（signature sequence）形成ATP结合袋（ATP-binding pockets,ABPs）ABP1和ABP2。ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用。ABP2由NBD2上的WalkA和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象。有一些CFrR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路。     

Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.     

Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.     

Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.     

The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.     

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.     

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.     

Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.     

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.     

Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.     

Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.     

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.     

On the mechanism of MgATP-dependent gating of CFTR Cl- channels

CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.     

Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.     

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.     

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.