Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Research Articles

CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel protein that is defective in individuals with cystic fibrosis (CF). To advance the rational design of CF therapies, it is important to elucidate how mutational defects in CFTR lead to its impairment and how pharmacological compounds interact with and alter CFTR. Here, using a helical-hairpin construct derived from CFTR's transmembrane (TM) helices 3 and 4 (TM3/4) and their intervening loop, we investigated the structural effects of a patient-derived CF-phenotypic mutation, E217G, located in the loop region of CFTR's membrane-spanning domain. Employing a single-molecule FRET assay to probe the folding status of reconstituted hairpins in lipid bilayers, we found that the E217G hairpin exhibits an altered adaptive packing behavior stemming from an additional GXXXG helix-helix interaction motif created in the mutant hairpin. This observation suggested that the misfolding and functional defects caused by the E217G mutation arise from an impaired conformational adaptability of TM helical segments in CFTR. The addition of the small-molecule corrector Lumacaftor exerts a helix stabilization effect not only on the E217G mutant hairpin, but also on WT TM3/4 and other mutations in the hairpin. This finding suggests a general mode of action for Lumacaftor through which this corrector efficiently improves maturation of various CFTR mutants.

On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.

Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.

Functional architecture of the CFTR chloride channel

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl(-) channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl(-) movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl(-) channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the three-dimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.

Declaration of Independence by CFTR's Transmembrane Domains

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily in that it functions as an ATP-gated chloride channel. Recent cysteine scanning studies in our lab demonstrate that both the sixth transmembrane segment (TM6) and its C-terminal counterpart, TM12, play pivotal roles in chloride permeation and are also involved in gating conformational changes. Here, to study the functional role of TM1 in gating and permeation, we introduced cysteine residues into this TM and assessed their reactivity towards internally-applied thiol-directed methanethiosulfonate (MTS) reagents. Our initial cysteine scanning experiments (1-2 patches for each cysteine-substituted cysless-CFTR channels) identified four positive hits, including E92, K95, Q98 and L102, where the negatively-charged MTSES reacting with engineered cysteines diminishes ATP-induced current. Surprisingly, according to the CFTR topology based on hydropathy plots, all these positions reside in the external half of TM1- contrary to results with TM6 and TM12. We also found that modification by the positively-charged MTSET enhances macroscopic current in K95C-, Q98C-, and L102C-cysless-CFTR channels. Intriguingly, at the single-channel level, we observed that whereas the mutation L102C appears to destabilize the gate, deposition of the MTSET adduct decreases the single-channel current amplitude but increases the Po. Moreover, channel gating persisted even after a complete removal of ATP, raising the possibility that gating motion in the TMDs of modified L102C channels is independent of that in their NBDs. Finally, MTSET modification of L102C is state-dependent, meaning that L102C-cysless-CFTR channels do not respond to the treatment of MTSET in the absence of ATP. These findings suggest that either position 102 moves during gating or gating motion in other regions of CFTR alters its reactivity towards MTSET. Overall, our preliminary data reveal several interesting yet enigmatic aspects of TM1 that call for further studies.

Conversion of an Alpha-Helical to a Beta-Sheet Membrane Protein Fold through Pro-Gly Mutations in the Loop of a Human CFTR Transmembrane Hairpin

Membrane proteins adopt two fundamental types of fold in nature. Eukaryotic membranes harbor only α-helical bundles composed of series of interacting hydrophobic α-helices separated by aqueous-located loops of varying length, while β-barrel structures are found in the outer membrane of gram-negative bacteria, mitochondria and chloroplasts. Their interwoven β-sheet structures are comprised of pairs of membrane-spanning β-strand hairpins with a characteristic alternating hydrophobic/small residue sequence pattern, connected by a short intervening loop. We report that mutations in the loop of a eukaryotic α-helical hairpin peptide from the cystic fibrosis transmembrane conductance regulator (CFTR) can induce the peptide to adopt a β-sheet structure in a membrane-mimetic environment. We characterized in sodium dodecylsulfate (SDS), sodium perfluorooctanoate (SPFO) and dodecyl maltoside (DDM) micelles several constructs of the CFTR transmembrane 3 and 4 (TM3-loop-TM4) ‘hairpins’ in which Pro-Gly residues have been either inserted or substituted to promote a β-turn within the putative loop region. Suitable positioning of the Pro-Gly couplet caused the formation of a ladder of discrete oligomers on SDS-PAGE and SPFO-PAGE. Circular dichroism spectroscopy showed that mutants displaying oligomeric patterns adopt a stable β-sheet structure in detergents. In addition, size-exclusion chromatography and pyrene excimer fluorescence analysis evidenced that β-sheet oligomers are not proliferative and/or random aggregates. Such a facile change from an α-helical to an oligomeric β-hairpin structure may have implications into the evolutionary connection between α-helical and β-barrel membrane proteins. Indeed, considering that the first membrane proteins were helical hairpins (1), these results could reproduce an early differentiation event in the evolution of membrane proteins.1. Frishman, D. (2010) Structural bioinformatics of membrane proteins, 1st ed., pp xi, 281 p. Springer, New York.

Misfolding of the Cystic Fibrosis Transmembrane Conductance Regulator and Disease

Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

Positional dependence of non-native polar mutations on folding of CFTR helical hairpins

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix–loop–helix constructs) which we term ‘helical hairpins’; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077–8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a 15N, 13C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of 15N and 1H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.

Role of the Extracellular Loop in the Folding of a CFTR Transmembrane Helical Hairpin

The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.

P97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation.

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.

Interhelical hydrogen bonds in the CFTR membrane domain.

Critical mutations in the membrane-spanning domains of proteins cause many human diseases. We report the expression in Escherichia coli of helix-loop-helix segments of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel domain in milligram quantities. Analysis of gel migration patterns of these constructs, in conjunction with circular dichroism spectroscopy, demonstrate that a neutral-to-charged, CF-phenotypic point mutation of a hydrophobic residue (V232D) in the CFTR transmembrane (TM) helix 4 induces a hydrogen bond with neighboring wild type Gln 207 in TM helix 3. As an electrostatic crosslink within a hydrocarbon phase, such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport. Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains.

Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane-spanning segment

The gene responsible for cystic fibrosis encodes a membrane protein – the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR) – in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3–4, a putative `helical hairpin' portion of the CFTR TM1–6 domain. The TM segment 3–4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3–4). Monitoring of the folding properties and conformational state(s) of the TM3–4 polypeptide using circular dichroism spectroscopy indicated a partial α-helical conformation in aqueous buffer, with up to 30% increase in α-helical content observed in membrane-mimetic environments.