Novel gain-of-function mutants identify a critical region within CFTR membrane-spanning domain 2 controlling cAMP-dependent and ATP-independent channel activation.

CFTR is an anion channel that has evolved from the mold of an ABC transporter. It possesses specific structural features, including a lateral portal between the cytoplasmic extensions of its transmembrane helices TM4 and TM6. This TM4-TM6 portal is lined by basic residues attracting anions from the cytosol towards the intracellular vestibule. Even though a symmetric, open portal is not observed at the level of the TM10/TM12 interface, basic amino acids are also present at this level, exposed to solvent in the vicinity of the regulatory R region, whose phosphorylation enables channel activation. Here, using all-atom molecular dynamics simulations in combination with functional and biochemical assays, we investigate the importance of these basic amino acids (R1158 and R1030), and of a neighboring aromatic amino acid (W846) in the regulation of CFTR activity. Results indicate that mutation of these amino acids globally increased channel activity and enabled channel opening by potentiators without the need to elevate cAMP levels. These effects (i) were observed even when the binding site of the potentiator VX-770 was mutated, revealing a probable independent mechanism, and (ii) were additive to one gain-of-function mutant within the selectivity filter. Taken together, our results indicate that the region of the membrane-spanning domain 2 (MSD2), symmetric to the lateral portal located between MSD1 TM4 and TM6, is a novel critical actor of CFTR regulation.

Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore.

Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.

The HERCulean task of recognizing, ubiquitinating, and shielding misfolded integral membrane proteins.

During ER-associated decay, unfolded membrane-resident proteins are targeted for removal and degradation by ubiquitin ligases whose identities and precise operations remain unclear. In this issue, Guerriero and Brodsky discuss new results from Kamada et al. (https://doi.org/10.1083/jcb.202308003) showing the clearance of misfolded CFTR by the E3 ligase HERC3.

The solute carrier family 26 member 9 modifies rapidly progressing cystic fibrosis associated with homozygous F508del CFTR mutation.

BACKGROUND AND AIMS: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations to the CF transmembrane conductance regulator (CFTR). Symptoms and severity of the disease can be quite variable suggesting modifier genes play an important role. MATERIALS AND METHODS: Exome sequencing was performed on six individuals carrying homozygous deltaF508 for CFTR genotype but present with rapidly progressing CF (RPCF). Data was analyzed using an unbiased genome-wide genetic burden test against 3076 controls. Single cell RNA sequencing data from LungMAP was utilized to evaluate unique and co-expression of candidate genes, and structural modeling to evaluate the deleterious effects of identified candidate variants. RESULTS: We have identified solute carrier family 26 member 9 (SLC26A9) as a modifier gene to be associated with RPCF. Two rare missense SLC26A9 variants were discovered in three of six individuals deemed to have RPCF: c.229G > A; p.G77S (present in two patients), and c.1885C > T; p.P629S. Co-expression of SLC26A9 and CFTR mRNA is limited across different lung cell types, with the highest level of co-expression seen in human (6.3 %) and mouse (9.0 %) alveolar type 2 (AT2) cells. Structural modeling suggests deleterious effects of these mutations as they are in critical protein domains which might affect the anion transport capability of SLC26A9. CONCLUSION: The enrichment of rare and potentially deleterious SLC26A9 mutations in patients with RPCF suggests SLC26A9 may act as an alternative anion transporter in CF and is a modifier gene associated with this lung phenotype.

Investigation of F508del CFTR unfolding and a search for stabilizing small molecules.

Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological temperatures that is rapidly degraded in the cell. This mutation is present in about 90% of cystic fibrosis patients, hence there is great interest in compounds reversing its instability. We have previously reported the expression of the mutated protein at low temperature and its purification in detergent. Here we describe the use of the protein to screen compounds present in a library of Federal Drug Administration (FDA) - approved drugs and also in a small natural product library. The kinetics of unfolding of F508del CFTR at 37 °C were probed by the increase in solvent-exposed cysteine residues accessible to a fluorescent reporter molecule. This occurred in a bi-exponential manner with a major (≈60%) component of half-life around 5 min and a minor component of around 60 min. The faster kinetics match those observed for loss of channel activity of F508del CFTR in cells at 37 °C. Most compounds tested had no effect on the fluorescence increase, but some were identified that significantly slowed the kinetics. The general properties of these compounds, and any likely mechanisms for inducing stability in purified CFTR are discussed. These experimental data may be useful for artificial intelligence - aided design of CFTR-specific drugs and in the identification of stabilizing additives for membrane proteins (in general).

Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Trivalent anions as probes of the CFTR channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel uses positively charged amino-acid side-chains to form binding sites for permeating anions. These binding sites have been investigated experimentally using a number of anionic probes. Mutations that alter the distribution of positive and negative charges within the pore have differential effects on the binding of monovalent versus divalent anions. This study uses patch clamp recording from wild-type and pore-mutant forms of CFTR to investigate small trivalent anions (Co(NO2)63-, Co(CN)3- and IrCl63-) as potential probes of anion binding sites. These anions caused weak block of Cl- permeation in wild-type CFTR (Kd ≥ 700 µM) when applied to the intracellular side of the membrane. Mutations that increase the density of positive charge within the pore (E92Q, I344K, S1141K) increased the binding affinity of these anions 80-280-fold, and also greatly increased the voltage-dependence of block, consistent with fixed charges in the pore affecting monovalent : multivalent anion selectivity. However, high-affinity pore block by Co(NO2)63-apparently did not alter channel gating, a hallmark of high-affinity binding of divalent Pt(NO2)42- ions within the pore. This work increases the arsenal of probes available to investigate anion binding sites within Cl- channel pores.

Analysis of AlphaMissense data in different protein groups and structural context.

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of missense variants. Since heterogenous performance on different types of proteins can be expected, we assessed the efficacy of AlphaMissense across several protein groups (e.g. soluble, transmembrane, and mitochondrial proteins) and regions (e.g. intramembrane, membrane interacting, and high confidence AlphaFold segments) using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on disordered datasets and ClinVar data related to the CFTR ABC protein. However, a superior performance was shown when benchmarked against the high quality CFTR2 database. Our results with CFTR emphasizes AlphaMissense's potential in pinpointing functional hot spots, with its performance likely surpassing benchmarks calculated from ClinVar and ProteinGym datasets.

The Folding Pathway of ABC Transporter CFTR: Effective and Robust.

De novo protein folding into a native three-dimensional structure is indispensable for biological function, is instructed by its amino acid sequence, and occurs along a vectorial trajectory. The human proteome contains thousands of membrane-spanning proteins, whose biosynthesis begins on endoplasmic reticulum-associated ribosomes. Nearly half of all membrane proteins traverse the membrane more than once, including therapeutically important protein families such as solute carriers, G-protein-coupled receptors, and ABC transporters. These mediate a variety of functions like signal transduction and solute transport and are often of vital importance for cell function and tissue homeostasis. Missense mutations in multispan membrane proteins can lead to misfolding and cause disease; an example is the ABC transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Even though our understanding of multispan membrane-protein folding still is rather rudimental, the cumulative knowledge of 20 years of basic research on CFTR folding has led to development of drugs that modulate the misfolded protein. This has provided the prospect of a life without CF to the vast majority of patients. In this review we describe our understanding of the folding pathway of CFTR in cells, which is modular and tolerates many defects, making it effective and robust. We address how modulator drugs affect folding and function of CFTR, and distinguish protein stability from its folding process. Since the domain architecture of (mammalian) ABC transporters are highly conserved, we anticipate that the insights we discuss here for folding of CFTR may lay the groundwork for understanding the general rules of ABC-transporter folding.

A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

Role of Hydrophobic Amino-Acid Side-Chains in the Narrow Selectivity Filter of the CFTR Chloride Channel Pore in Conductance and Selectivity.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Structural analysis of CFTR has identified a narrow, hydrophobic region close to the extracellular end of the open channel pore that may function as a selectivity filter. The present study combines comprehensive mutagenesis of hydrophobic amino-acid side-chains within the selectivity filter with functional evaluation of channel Cl- conductance and anion selectivity. Among these hydrophobic amino-acids, one (F337) appears to play a dominant role in determining both conductance and selectivity. Anion selectivity appears to depend on both side-chain size and hydrophobicity at this position. In contrast, conductance is disrupted by all F337 mutations, suggesting that unique interactions between permeating Cl- ions and the native phenylalanine side-chain are important for conductance. Surprisingly, a positively charged lysine side-chain can be substituted for several hydrophobic residues within the selectivity filter (including F337) with only minor changes in pore function, arguing against a crucial role for overall hydrophobicity. These results suggest that localized interactions between permeating anions and amino-acid side-chains within the selectivity filter may be more important in determining pore functional properties than are global features such as overall hydrophobicity.

CFTR Folding: From Structure and Proteostasis to Cystic Fibrosis Personalized Medicine.

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations in the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR). Class-II mutants of CFTR lack intermolecular interactions important for CFTR structural stability and lead to misfolding. Misfolded CFTR is detected by a diverse suite of proteostasis factors that preferentially bind and route mutant CFTR toward premature degradation, resulting in reduced plasma membrane CFTR levels and impaired chloride ion conductance associated with CF. CF treatment has been vastly improved over the past decade by the availability of small molecules called correctors. Correctors directly bind CFTR, stabilize its structure by conferring thermodynamically favorable interactions that compensate for mutations, and thereby lead to downstream folding fidelity. However, each of over 100 Class-II CF causing mutations causes unique structural defects and shows a unique response to drug treatment, described as theratype. Understanding CFTR structural defects, the proteostasis factors evaluating those defects, and the stabilizing effects of CFTR correctors will illuminate a path toward personalized medicine for CF. Here, we review recent advances in our understanding of CFTR folding, focusing on structure, corrector binding sites, the mechanisms of proteostasis factors that evaluate CFTR, and the implications for CF personalized medicine.

CFTR function, pathology and pharmacology at single-molecule resolution.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Optimization of CFTR gating through the evolution of its extracellular loops.

CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). CFTR is activated by phosphorylation, and phosphorylated channels exhibit "bursting" behavior-"bursts" of openings separated by short "flickery" closures and flanked by long "interburst" closures-driven by ATP binding/hydrolysis at two nucleotide-binding domains. The human channel (hCFTR) and the distant zebrafish ortholog (zCFTR) display differences both in their gating properties and structures. In phosphorylated ATP-bound hCFTR, the hR117 side chain, conserved across evolution, forms an H-bond that stabilizes the open state. Lack of that bond in the hR117H mutant causes CF. In the phosphorylated ATP-bound zCFTR structure that H-bond is not observable. Here, we show that the zR118H mutation does not affect the function of zCFTR. Instead, we identify an H-bond between the zS109 and zS120 side chains of phosphorylated ATP-bound, but not of unphosphorylated apo-, zCFTR. We investigate the role of that interaction using thermodynamic mutant cycles built on gating parameters determined in inside-out patch clamp recordings. We find that zS109 indeed forms an H-bond with zN120 in the flickery closed state, but not in the open or interburst closed states. Although in hCFTR an isoleucine (hI119) replaces the asparagine, mutation hS108A produces a strong hR117H-like phenotype. Since the effects of the latter two mutations are not additive, we conclude that in hCFTR these two positions interact, and the hS108-hR117 and hR117-hE1124 H-bonds cooperate to stabilize the open state. These findings highlight an example of how the gating mechanism was optimized during CFTR molecular evolution.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

A conserved WXXE motif is an apical delivery determinant of ABC transporter C subfamily isoforms.

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl- excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41-60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4.

Role of inhaled antibiotics in the era of highly effective CFTR modulators.

Recurrent and chronic bacterial infections are common in people with cystic fibrosis (CF) and contribute to lung function decline. Antibiotics are the mainstay in the treatment of exacerbations and chronic bacterial infection in CF. Inhaled antibiotics are effective in treating chronic respiratory bacterial infections and eradicating Pseudomonas aeruginosa from the respiratory tract, with limited systemic adverse effects. In the past decade, highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have become a new therapy that partially corrects/opens chloride transport in patients with selected CFTR mutations, restoring mucus hydration and improving mucociliary clearance. The recent triple CFTR modulator combination is approved for ∼80-90% of the CF population and significantly reduces pulmonary exacerbations and improves respiratory symptoms and lung function. CFTR modulators have shifted the focus from symptomatic treatment to personalised/precision medicine by targeting genotype-specific CFTR defects. While these are highly effective, they do not fully normalise lung physiology, stop inflammation or resolve chronic lung damage, such as bronchiectasis. The impact of these new drugs on lung health is likely to change the future management of chronic pulmonary infections in people with CF. This article reviews the role of inhaled antibiotics in the era of CFTR modulators.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

Molecular structures reveal synergistic rescue of Δ508 CFTR by Trikafta modulators.

The predominant mutation causing cystic fibrosis, a deletion of phenylalanine 508 (Δ508) in the cystic fibrosis transmembrane conductance regulator (CFTR), leads to severe defects in CFTR biogenesis and function. The advanced therapy Trikafta combines the folding corrector tezacaftor (VX-661), the channel potentiator ivacaftor (VX-770), and the dual-function modulator elexacaftor (VX-445). However, it is unclear how elexacaftor exerts its effects, in part because the structure of Δ508 CFTR is unknown. Here, we present cryo-electron microscopy structures of Δ508 CFTR in the absence and presence of CFTR modulators. When used alone, elexacaftor partially rectified interdomain assembly defects in Δ508 CFTR, but when combined with a type I corrector, did so fully. These data illustrate how the different modulators in Trikafta synergistically rescue Δ508 CFTR structure and function.

Triangulating variation in the population to define mechanisms for precision management of genetic disease.

To understand mechanistically how the protein fold is shaped by therapeutics to inform precision management of disease, we developed variation-capture (VarC) mapping. VarC triangulates sparse sequence variation information found in the population using Gaussian process regression (GPR)-based machine learning to define the combined pairwise-residue interactions contributing to dynamic protein function in the individual in response to therapeutics. Using VarC mapping, we now reveal the pairwise-residue covariant relationships across the entire protein fold of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) to define the molecular mechanisms of clinically approved CF chemical modulators. We discover an energetically destabilized covariant core containing a di-acidic YKDAD endoplasmic reticulum (ER) exit code that is only weakly corrected by current therapeutics. Our results illustrate that VarC provides a generalizable tool to triangulate information from genetic variation in the population to mechanistically discover therapeutic strategies that guide precision management of the individual.