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.

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 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

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

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.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Learning the ABCs one at a time: structure and mechanism of ABC transporters

2019 ◽  
Vol 47 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Robert C. Ford ◽  
Konstantinos Beis
Keyword(s):  
Abc Transporters ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Bacterial Cells ◽  
The Novel ◽  
Mechanism Model ◽  
Binding Domains ◽  
Atp Binding And Hydrolysis ◽  
Alternating Access

Abstract ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.

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 Identification of a novel post-hydrolytic state in CFTR gating

2012 ◽  
Vol 139 (5) ◽  
pp. 359-370 ◽  
Author(s):  
Kang-Yang Jih ◽  
Yoshiro Sohma ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Free Energy ◽  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Fast Response ◽  
Structural Basis ◽  
Atp Binding ◽  
Closed State ◽  
Binding Domains ◽  
Abc Protein

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

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 Modulation of CFTR gating by permeant ions

2014 ◽  
Vol 145 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Han-I Yeh ◽  
Jiunn-Tyng Yeh ◽  
Tzyh-Chang Hwang
Keyword(s):  
Atp Hydrolysis ◽  
Single Channel ◽  
Common Mechanism ◽  
Transmembrane Conductance Regulator ◽  
Open Probability ◽  
Binding Domains ◽  
Gating Model ◽  
Sites Of Action ◽  
Opening And Closing ◽  
Closing Rate

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR’s transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

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.

Stimulatory and Inhibitory Functions of the R Domain on CFTR Chloride Channel

Physiology ◽  
2000 ◽  
Vol 15 (3) ◽  
pp. 154-158 ◽  
Author(s):  
Jianjie Ma
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Chloride Channel ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing ◽  
Gating Process ◽  
Kinase A ◽  
R Domain

CFTR is a chloride channel whose gating process involves coordinated interactions among the regulatory (R) domain and the nucleotide-binding folds (NBFs). Protein kinase A phosphorylation of serine residues renders the R domain from inhibitory to stimulatory and enables ATP binding and hydrolysis at the NBFs, which in turn control opening and closing of the chloride channel.

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