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

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 â€œpartialâ€ 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.

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

Journal of Biological Chemistry ◽

10.1074/jbc.m110.172817 ◽

2010 ◽

Vol 285 (48) ◽

pp. 37663-37671 ◽

Cited By ~ 13

Author(s):

Ming-Feng Tsai ◽

Kang-Yang Jih ◽

Hiroyasu Shimizu ◽

Min Li ◽

Tzyh-Chang Hwang

Keyword(s):

Cystic Fibrosis ◽

Binding Site ◽

Atp Binding ◽

Atp Binding Site ◽

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

Journal of Biological Chemistry ◽

10.1074/jbc.m609942200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4533-4544 ◽

Cited By ~ 71

Author(s):

Wei Wang ◽

Karen Bernard ◽

Ge Li ◽

Kevin L. Kirk

Keyword(s):

Cystic Fibrosis ◽

Nucleotide Binding ◽

Salt Transport ◽

Atp Binding ◽

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.

ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2008.0191 ◽

2008 ◽

Vol 364 (1514) ◽

pp. 247-255 ◽

Cited By ~ 32

Author(s):

Daniella Muallem ◽

Paola Vergani

Keyword(s):

Cystic Fibrosis ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Atp Binding ◽

Functional Difference ◽

Cellular Functions ◽

Binding Domains ◽

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.

Control of the CFTR channel's gates

Biochemical Society Transactions ◽

10.1042/bst0331003 ◽

2005 ◽

Vol 33 (5) ◽

pp. 1003-1007 ◽

Cited By ~ 13

Author(s):

P. Vergani ◽

C. Basso ◽

M. Mense ◽

A.C. Nairn ◽

D.C. Gadsby

Keyword(s):

Cystic Fibrosis ◽

Ion Channel ◽

Single Channel ◽

Atp Binding ◽

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201110608 ◽

2011 ◽

Vol 137 (6) ◽

pp. 549-562 ◽

Cited By ~ 31

Author(s):

Andras Szollosi ◽

Daniella R. Muallem ◽

László Csanády ◽

Paola Vergani

Keyword(s):

Atp Binding ◽

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.

The Two ATP Binding Sites of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Play Distinct Roles in Gating Kinetics and Energetics

The Journal of General Physiology ◽

10.1085/jgp.200609622 ◽

2006 ◽

Vol 128 (4) ◽

pp. 413-422 ◽

Cited By ~ 53

Author(s):

Zhen Zhou ◽

Xiaohui Wang ◽

Hao-Yang Liu ◽

Xiaoqin Zou ◽

Min Li ◽

...

Keyword(s):

Cystic Fibrosis ◽

Binding Sites ◽

Aromatic Amino Acids ◽

Atp Binding ◽

Binding Domains ◽

Adenine Ring ◽

Transporter Family ◽

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.

Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

The Journal of General Physiology ◽

10.1085/jgp.200810049 ◽

2008 ◽

Vol 133 (1) ◽

pp. 69-77 ◽

Cited By ~ 6

Author(s):

Xiaohui Wang ◽

Silvia G. Bompadre ◽

Min Li ◽

Tzyh-Chang Hwang

Keyword(s):

Cystic Fibrosis ◽

Chloride Channel ◽

Binding Pocket ◽

Cysteine Residue ◽

Atp Binding ◽

Binding Domains ◽

Signature Sequence ◽

The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd2+] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd2+ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd2+. On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Collectively, these results suggest that the effect of Cd2+ is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

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

Biochemical Journal ◽

10.1042/bj20081068 ◽

2008 ◽

Vol 416 (1) ◽

pp. 129-136 ◽

Cited By ~ 14

Author(s):

Luba Aleksandrov ◽

Andrei Aleksandrov ◽

John R. Riordan

Keyword(s):

Atp Hydrolysis ◽

Nucleotide Binding ◽

Binding Pocket ◽

Atp Binding ◽

Bivalent Cation ◽

Binding Domains ◽

Rapid Hydrolysis ◽

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.

Progress in precision medicine in cystic fibrosis: a focus on CFTR modulator therapy

Breathe ◽

10.1183/20734735.0112-2021 ◽

2021 ◽

Vol 17 (4) ◽

pp. 210112

Author(s):

Daniel H. Tewkesbury ◽

Rebecca C. Robey ◽

Peter J. Barry

Keyword(s):

Cystic Fibrosis ◽

Precision Medicine ◽

Real World ◽

Chloride Ion ◽

Therapeutic Approaches ◽

Cftr Protein ◽

Cystic Fibrosis Transmembrane ◽

Cftr Modulators

The genetic multisystem condition cystic fibrosis (CF) has seen a paradigm shift in therapeutic approaches within the past decade. Since the first clinical descriptions in the 1930s, treatment advances had focused on the downstream consequences of a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel. The discovery of the gene that codes for CFTR and an understanding of the way in which different genetic mutations lead to disruption of normal CFTR function have led to the creation and subsequent licensing of drugs that target this process. This marks an important move towards precision medicine in CF and results from clinical trials and real-world clinical practice have been impressive. In this review we outline how CFTR modulator drugs restore function to the CFTR protein and the progress that is being made in this field. We also describe the real-world impact of CFTR modulators on both pulmonary and multisystem complications of CF and what this will mean for the future of CF care.