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 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.

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

2008 ◽  
Vol 133 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Xiaohui Wang ◽  
Silvia G. Bompadre ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Binding Pocket ◽  
Cysteine Residue ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Signature Sequence ◽  
Cystic Fibrosis Transmembrane

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.

scholarly journals Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein

1999 ◽  
Vol 340 (1) ◽  
pp. 227-235 ◽  
Author(s):  
Christoph RANDAK ◽  
Ennes A. AUERSWALD ◽  
Irmgard ASSFALG-MACHLEIDT ◽  
William W. REENSTRA ◽  
Werner MACHLEIDT
Keyword(s):  
Cystic Fibrosis ◽  
Adenylate Kinase ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Enhanced Fluorescence ◽  
Chloride Currents ◽  
Cystic Fibrosis Transmembrane ◽  
Ordered State

In the presence of ATP, genistein, like the ATP analogue adenosine 5ʹ-[β,γ-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 μM and 63 μM for ATPase, 15 μM and 54 μM for GTPase, and 46 μM and 142 μM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.

scholarly journals Perturbation of the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibits Its ATPase Activity

2000 ◽  
Vol 276 (15) ◽  
pp. 11575-11581 ◽  
Author(s):  
Ilana Kogan ◽  
Mohabir Ramjeesingh ◽  
Ling-Jun Huan ◽  
Yanchun Wang ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Cross Talk ◽  
Nucleotide Binding ◽  
Cystic Fibrosis Gene ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl−) flux in affected epithelial tissues. CFTR is a Cl−channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl−permeates. Mutations of residues lining the channel pore (e.g.R347D) are typically thought to cause disease by altering the interaction of Cl−with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in thein vivoregulation of CFTR in health and disease.

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