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.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.     

Functional dissection of the R domain of cystic fibrosis transmembrane conductance regulator

Exogenously expressed unphosphorylated sub-domains of the R domain block CFTR Cl- channels in the planar lipid bilayer, though the block differs from block with full length R domain. Full length R domain peptide (aa 588-855) blocks CFTR Cl- channels quickly, completely and permanently. Two sub-domains, RD1RD2 (aa 588-805) and RD2TM (aa 672-855), also inhibit CFTR Cl- channels, but the block takes longer to effect and is not complete. Shorter sequences, RD1 (aa 588-746) and RD2 (aa 672-805), fail to effect any block. These data suggest that either the amino-terminal or carboxy-terminal portions of the R domain protein or its stabilized secondary structure are critical to functional regulation.     

Partial purification of the cystic fibrosis transmembrane conductance regulator.

We have investigated several purification strategies for the cystic fibrosis transmembrane regulator (CFTR) based on its structural similarity to other proteins of the traffic ATPase/ABC transporter family. Recombinant CFTR expressed in heterologous cells was readily solubilized by digitonin and initially separated from the majority of other cellular proteins by sucrose density gradient centrifugation. CFTR, with two predicted nucleotide binding domains, bound avidly to several triazine dye columns, although elution with MgATP, MgCl2, or high ionic strength buffers was inefficient. CFTR did not bind to either ATP or ADP coupled to agarose. Because CFTR is a glycoprotein we investigated its binding to lectin columns. CFTR bound readily to wheat germ agglutinin, but poorly to Lens culinaris agglutinin. CFTR was enriched 9-10 times when eluted from wheat germ agglutinin with N-acetylglucosamine. This enrichment was tripled if lectin chromatography followed sucrose gradient centrifugation. Our results suggest the combination of sucrose density gradient centrifugation and lectin chromatography would be a satisfactory approach to initial purification of CFTR expressed in heterologous cells.     

Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.     

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.     

Destabilization of the transmembrane domain induces misfolding in a phenotypic mutant of cystic fibrosis transmembrane conductance regulator

Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses approximately 50% helicity, implying a membrane insertion defect in the latter mutant. A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. The correlated in vitro/in vivo results demonstrate that destabilizing local hydrophobic character represents a sufficient signal for marking CFTR as a non-native protein by the ER quality control, with accompanying deleterious consequences to global protein folding events.     

Glycosylation and the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.     

Domain-domain associations in cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is caused by mutations inthe cystic fibrosis transmembrane conductance regulator (CFTR) gene.CFTR is a chloride channel whose activity requires protein kinaseA-dependent phosphorylation of an intracellular regulatory domain(R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs).To identify potential sites of domain-domain interaction within CFTR,we expressed, purified, and refolded histidine (His)- andglutathione-S-transferase (GST)-tagged cytoplasmic domainsof CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated bymeasuring tryptophan fluorescence quenching. Trypticdigestion of in vitro phosphorylated his-NBD1-R and in situphosphorylated CFTR generated the same phosphopeptides. An interactionbetween NBD1-R and NBD2 was assayed by tryptophan fluorescencequenching. Binding among all pairwise combinations of R-domain, NBD1,and NBD2 was demonstrated with an overlay assay. To identifyspecific sites of interaction between domains of CFTR, an overlay assaywas used to probe an overlapping peptide library spanning allintracellular regions of CFTR with his-NBD1, his-NBD2, andGST-R-domain. By mapping peptides from NBD1 and NBD2 that bound toother intracellular domains onto crystal structures for HisP, MalK, andRad50, probable sites of interaction between NBD1 and NBD2 wereidentified. Our data support a model where NBDs form dimers with theATP-binding sites at the domain-domain interface.

     

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in ΔF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.     

Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembrane conductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number of exocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CF phenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations have afforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and have allowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhanced understanding of the role of CFTR in regulating epithelial ion transport, have led to the development of therapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy, termed 'protein repair therapy', is aimed at improving the regulation of epithelial ion transport by mutant CFTRs in a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction, yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations. Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtain clinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold great promise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.     

An unstable transmembrane segment in the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.     

Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.     

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.     

Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

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 gamma-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.