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

2011 ◽  
Vol 137 (6) ◽  
pp. 549-562 ◽  
Author(s):  
Andras Szollosi ◽  
Daniella R. Muallem ◽  
László Csanády ◽  
Paola Vergani
Keyword(s):  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Gating Model ◽  
Channel Opening ◽  
Independent Technique ◽  
Atp Binding And Hydrolysis ◽  
Thermodynamic Coupling

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.

scholarly journals Functional and pharmacological importance of the composite ATP binding site 1 in CFTR chloride channels

2010 ◽  
Author(s):  
◽  
Ming-Feng Tsai
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Molecular Model ◽  
Chloride Ion ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Opening And Closing

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel whose defects cause the deadly genetic disease cystic fibrosis (CF). Like other ATP binding cassette (ABC) proteins, CFTR encompasses two cytoplasmic nucleotide binding domains (NBDs). Upon ATP binding, the two NBDs can coalesce into a head-to-tail dimer with ATP buried at two interfacial composite sites (sites 1 and 2). Although evidence suggests that gating of CFTR is mainly controlled by site 2, the role of site 1 remains less understood. I have used pyrophosphate as a probe or adopted a ligand exchange protocol to investigate ATP binding status in site 1 in real time. With these novel approaches, I have identified a “partial” NBD dimer state mediated by an ATP molecule tightly bound in site 1. A molecular model of CFTR gating was then established with opening and closing of CFTR coupled to the formation and partial separation of the NBD dimer. Moreover, I discovered several mutations that enhance ATP binding in site 1 and demonstrated that the activity of CF-associated mutant channels, Î"F508- and G551D-CFTR, can be significantly improved by these mutations, thus providing evidence that site 1 is a potential target for developing pharmaceutical reagents to treat patients with CF.

scholarly journals Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

2006 ◽  
Vol 282 (7) ◽  
pp. 4533-4544 ◽  
Author(s):  
Wei Wang ◽  
Karen Bernard ◽  
Ge Li ◽  
Kevin L. Kirk
Keyword(s):  
Cystic Fibrosis ◽  
Nucleotide Binding ◽  
Salt Transport ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Channel Opening ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

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.

Control of the CFTR channel's gates

2005 ◽  
Vol 33 (5) ◽  
pp. 1003-1007 ◽  
Author(s):  
P. Vergani ◽  
C. Basso ◽  
M. Mense ◽  
A.C. Nairn ◽  
D.C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channel ◽  
Single Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Binding Domains ◽  
Abc Protein ◽  
Channel Opening ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1–NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

scholarly journals Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

2010 ◽  
Vol 135 (5) ◽  
pp. 399-414 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Abc Proteins ◽  
Atp Binding Site ◽  
Functional Studies ◽  
Binding Domains ◽  
Gating Model ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

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

2008 ◽  
Vol 416 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Luba Aleksandrov ◽  
Andrei Aleksandrov ◽  
John R. Riordan
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bivalent Cation ◽  
Binding Domains ◽  
Rapid Hydrolysis ◽  
Cystic Fibrosis Transmembrane

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, Mg2+ 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 [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 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.

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

2015 ◽  
Vol 145 (4) ◽  
pp. 261-283 ◽  
Author(s):  
Luiz A. Poletto Chaves ◽  
David C. Gadsby
Keyword(s):  
Atp Hydrolysis ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Dimer Interface ◽  
Binding Domains ◽  
Channel Opening ◽  
Order Of Magnitude ◽  
Signature Sequence ◽  
Dependent Modification ◽  
Opening And Closing

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.

scholarly journals ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

2008 ◽  
Vol 364 (1514) ◽  
pp. 247-255 ◽  
Author(s):  
Daniella Muallem ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Functional Difference ◽  
Transmembrane Conductance Regulator ◽  
Cellular Functions ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

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

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

2014 ◽  
Vol 289 (44) ◽  
pp. 30364-30378 ◽  
Author(s):  
Wei Wang ◽  
Bryan C. Roessler ◽  
Kevin L. Kirk
Keyword(s):  
Electrostatic Interaction ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Charge Reversal ◽  
Allosteric Coupling ◽  
Binding Domains ◽  
Channel Opening ◽  
Open States ◽  
Intracellular Loops ◽  
R Domain

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.

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