Structure Of CFTR Research Articles

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.

290 Extending the success of elexacaftor-tezacaftor-ivacaftor to rare mutations—insights from CFTR structure and modulator binding

290 Extending the success of elexacaftor-tezacaftor-ivacaftor to rare mutations—insights from CFTR structure and modulator binding

WS09.01 Extending the success of Trikafta to rare mutations - insights from CFTR structure and modulator binding

ETI offers therapeutic opportunities for most CF patients but not for many with rare mutations. We established patient-derived organoids with the Q1100P mutation, not reported in CFTR2. We obtained biopsies from 3 patients heterozygous for Q1100P and a stop mutation (C225X, likely nonresponsive to ETI) or a missense (K163E). We investigated CFTR function after treatment with modulators.

Structure basis of CFTR folding, function and pharmacology

The root cause of cystic fibrosis (CF), the most common life-shortening genetic disease in the Caucasian population, is the loss of function of the CFTR protein, which serves as a phosphorylation-activated, ATP-gated anion channel in numerous epithelia-lining tissues. In the past decade, high-throughput drug screening has made a significant stride in developing highly effective CFTR modulators for the treatment of CF. Meanwhile, structural-biology studies have succeeded in solving the high-resolution three-dimensional (3D) structure of CFTR in different conformations. Here, we provide a brief overview of some striking features of CFTR folding, function and pharmacology, in light of its specific structural features within the ABC-transporter superfamily. A particular focus is given to CFTR's first nucleotide-binding domain (NBD1), because folding of NBD1 constitutes a bottleneck in the CFTR protein biogenesis pathway, and ATP binding to this domain plays a unique role in the functional stability of CFTR. Unraveling the molecular basis of CFTR folding, function, and pharmacology would inspire the development of next-generation mutation-specific CFTR modulators.

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis

BackgroundIn cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation. MethodsGenetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated. ResultsOrganoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation. ConclusionsIn vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.

Genomic, transcriptomic, and protein landscape profile of CFTR and cystic fibrosis.

Cystic Fibrosis (CF) is caused most often by removal of amino acid 508 (Phe508del, deltaF508) within CFTR, yet dozens of additional CFTR variants are known to give rise to CF and many variants in the genome are known to contribute to CF pathology. To address CFTR coding variants, we developed a sequence-to-structure-to-dynamic matrix for all amino acids of CFTR using 233 vertebrate species, CFTR structure within a lipid membrane, and 20ns of molecular dynamic simulation to assess known variants from the CFTR1, CFTR2, ClinVar, TOPmed, gnomAD, and COSMIC databases. Surprisingly, we identify 18 variants of uncertain significance within CFTR from diverse populations that are heritable and a likely cause of CF that have been understudied due to nonexistence in Caucasian populations. In addition, 15 sites within the genome are known to modulate CF pathology, where we have identified one genome region (chr11:34754985-34836401) that contributes to CF through modulation of expression of a noncoding RNA in epithelial cells. These 15 sites are just the beginning of understanding comodifiers of CF, where utilization of eQTLs suggests many additional genomics of CFTR expressing cells that can be influenced by genomic background of CFTR variants. This work highlights that many additional insights of CF genetics are needed, particularly as pharmaceutical interventions increase in the coming years.

Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R.

Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells. Structural analyses were also performed to study the molecular environment of the W361 residue. Western blot showed that W361R-CFTR was not efficiently processed to a mature band C, similar to F508del CFTR, but unlike F508del CFTR, it did exhibit significant transport activity at the cell surface in response to cAMP agonists. Importantly, W361R-CFTR also responded well to CFTR modulators: its maturation defect was efficiently corrected by VX-809 treatment and its channel activity further potentiated by VX-770. Based on these results, we postulate that W361R is a novel class-2 CF mutation that causes abnormal protein maturation which can be corrected by VX-809, and additionally potentiated by VX-770, two FDA-approved small molecules. At the structural level, W361 is located within a class-2 CF mutation hotspot that includes other mutations that induce variable disease severity. Analysis of the 3D structure of CFTR within a lipid environment indicated that W361, together with other mutations located in this hotspot, is at the edge of a groove which stably accommodates lipid acyl chains. We suggest this lipid environment impacts CFTR folding, maturation and response to CFTR modulators.

CFTR structure, stability, function and regulation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.

CFTR structure: lassoing cystic fibrosis.

Loss of function of the CFTR anion channel leads to cystic fibrosis, the most common inherited condition in humans of European origin. A recently reported structure for CFTR at 3.7-A resolution reveals an unexpected 'lasso' domain and provides new insights into channel function in healthy individuals and in people with cystic fibrosis.

Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.

CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains.

Timing of CFTR Pore Opening and Structure of Its Transition State

In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrierfor opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structurepharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.

WS23.1 Models of the 3D structure of CFTR: from the understanding of the protein functions to the design of correctors

B. Hoffmann1, J.-P. Mornon1, B. Boucherle2, A. Fortune2, R. Haudecoeur2, C. Boinot3, M. Jollivet3, J.-L. Decout2, F. Becq3, P. Lehn4, I. Callebaut1. 1Universite Pierre et Marie Curie, CNRS, MNHN, IRD, IMPMC, UMR 7590, Paris, France; 2Universite Joseph Fourier, UJF/CNRS, DPM, UMR 5063, Grenoble, France; 3Universite de Poitiers, CNRS, Laboratoire STIM, ERL 7368, Poitiers, France; 4Universite de Bretagne Occidentale, INSERM U 1078, Brest, France

Managing the Underlying Cause of Cystic Fibrosis: A Future Role for Potentiators and Correctors

Cystic fibrosis (CF), a severe genetic disease, is caused by mutations that alter the structure and function of CFTR, a plasma membrane channel permeable to chloride and bicarbonate. Defective anion transport in CF irreversibly damages the lungs, pancreas, liver, and other organs. CF mutations cause loss of CFTR function in multiple ways. In particular, class 3 mutations such as p.Gly551Asp strongly decrease the time spent by CFTR in the open state (gating defect). Instead, class 2 mutations impair the maturation of CFTR protein and its transport from the endoplasmic reticulum to the plasma membrane (trafficking defect). The deletion of phenylalanine 508 (p.Phe508del), the most frequent mutation among CF patients (70-90 %), destabilizes the CFTR protein, thus causing both a trafficking and a gating defect. These two defects can be overcome with drug-like molecules generically called correctors and potentiators, respectively. The potentiator Kalydeco™ (also known as Ivacaftor or VX-770), developed by Vertex Pharmaceuticals, has been recently approved by the US FDA and the European Medicines Agency (EMA) for the treatment of CF patients carrying at least one CFTR allele with the p.Gly551Asp mutation (2-5 % of all patients). In contrast, the corrector VX-809, which significantly improves p.Phe508del-CFTR trafficking in vitro, is still under study in clinical trials. Because of multiple defects caused by the p.Phe508del mutation, it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action. This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect.

WS4.2 Exploration of the ATP binding site in CFTR-NBD1 for enhanced ligand binding

CFTR is a chloride channel belonging to the ABC superfamily. In previous work, we have developed models of the whole 3D structure of human CFTR based on experimental structures of proteins belonging to the ABC superfamily, in outward and inward-facing conformations (Sav1866, Msba) (Mornon 2008 & 2009, Serohijos 2008). These models, together with molecular dynamics (MD) simulations based on Sav1866 (Norimatsu 2012, Dalton 2012) and PgP (Furukawa 2013), were used to understand the molecular basis of the CFTR functioning. Here, we present a refined sequence alignment and a model of the CFTR open channel which uses information from all five known 3D structures of ABC exporters. This model provides a new vision of the pore and the interfaces of the intracellular loops 1 and 3 with the NBDs. We also describe a new model of CFTR based on the structure of the heterodimeric ABC transporter TM287-TM288 in an inward-facing state. As CFTR, this structure has nonequivalent ATP-binding sites. The two NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP-binding sites. Thus, this new model of the CFTR closed channel allows unprecedented insights into the role of specific amino acids, typifying the CFTR noncanonical ATP-binding site. MD has been performed in order to relax the model and obtain a structure probably closer to reality. The refined models presented here pave the way for a search of molecules able to fix the assembly of the protein and its thermal stability, a major cause of the low ability of CFTR to reach and stay at the plasma membrane. Supported by the French Association Vaincre La Mucoviscidose Paris.

Strategies to circumvent the CFTR defect in cystic fibrosis

Mutations within the gene encoding for the chloride ion channel CFTR results in cystic fibrosis, the most common autosomal recessive genetic disease in the Caucasian population. CFTR regulates absorption and secretion mechanisms across intestinal and airway mucosae. Although the intestinal phenotype can be clinically handled, chronic infection and inflammation of the lungs of CF patients remains the principal cause of morbidity and mortality. The aim of this Research Topic is to provide to the readers the most recent information available on “Strategies to circumvent the CFTR defect in cystic fibrosis.” The Research Topic is divided in three mains parts: the first part describes the CFTR structure, processing and regulation of the normal and mutant ion channels. Anna Patrick and Philip Thomas (University of Texas Southwestern Medical Center, Dallas, USA) review the molecular interactions leading to the complex folding process of the CFTR protein and how essential steps are disrupted in CFTR mutants. Soo Jung Kim and William Skach (Oregon Health and Science University, Portland, USA) detail the timing and coordination of specific folding steps of the native CFTR in and across the ER membrane. Colleen Weiler and Mitchell Drumm (Case Western Reserve University, Cleveland, USA) broaden our views with genetic studies that have identified variant genes implicated in the clinical manifestations of CF. The second part of the Special topics discusses the molecular targets to rescue CFTR processing. Rebecca Chanoux and Ronald Rubenstein describe the current knowledge of the network of cellular chaperones that facilitate the folding and trafficking of CFTR to the plasma membrane. Then, a series of papers by the groups of Teresinha Leal (Universite Catholique de Louvain, Brussels, Belgium) and John Hanrahan (McGill University, Montreal, Canada) report interesting observations regarding the potential of several inhibitors in correcting the CF phenotype at the level of ion transport and inflammation. Luigi Maiuri and collaborators (European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy) propose to target the intracellular environment in order to reestablish functional autophagy in CF epithelial cells. The third chapter is dedicated to new molecules that have already been developed or in development and able to rescue CFTR channel function by targeting mutant CFTR at the mRNA (Michael Wilschanski, Hadassah Hospitals- Hebrew University, Jerusalem, Israel) and/or at the protein (Nicoletta Pedemonte and Luis Galietta, Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini Genova, Italy) levels. Finally, the Special Topic is concluded by an in-depth review by Christine Bear and collaborators (The Hospital for Sick Children, Toronto, Canada). Importantly, the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development are discussed. We are convinced that we achieved a very interesting Special Topics thanks to the outstanding contributions of all authors. We are especially grateful to the authors for having believed in this project and accepted to share their knowledge. All the manuscripts have been peer-reviewed and we would like to thank the experts for helping us to reach an issue of high standard.

Molecular Dynamics Simulations of CFTR Gating and Conductance

The crystal structure of CFTR, the chloride ion channel defective in cystic fibrosis, has not yet been solved. Here we present two new homology models of human CFTR based on template structures from related ABC transporters. The first is based on the bacterial transporter Sav1866 and is representative of the open channel state of CFTR. Unlike previous Sav1866-based homology models of CFTR, our model incorporates several key structural features expected from experiment, including the proper positioning of pore-lining residues and important salt bridges. The second is based on the crystal structure of murine P-glycoprotein and models the closed state of CFTR. We performed targeted molecular dynamics simulations using these two models as end states, in order to gain insight into the conformational changes that CFTR undergoes during its gating cycle. Our simulations reveal that CFTR gating involves a conformational wave that is initiated at the nucleotide-binding domains, and propagates through interactions in the intracellular loops to the membrane-spanning domains. Analysis of our simulations also led to a better understanding of the relative motions of the twelve transmembrane helices in CFTR, and how they alter pore structure during gating. Our MD simulations allowed identification of key inter-residue interactions that stabilize the end states as well as transient interactions that may exist in the intermediate stages in gating. Charting the progression of these interactions provides a timeline of events likely to occur during gating, and may prove invaluable in furthering our understanding of structure-function relationships in CFTR. Finally, we report on preliminary simulations of chloride ion conduction in the open CFTR channel, which reveal key interactions of pore-lining residues with passing ions, as well as identifying the putative narrow region in the pore that may form a selectivity filter in this channel. (NIH-2R56DK056481-07)

Functional Rescue of F508del-CFTR Using Small Molecule Correctors

High-throughput screens for small molecules that are effective in “correcting” the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

48 Insights into the transmission interfaces of human CFTR from molecular modeling

Models of the 3D structure of full-length human CFTR described during the last years have provided new insights into the molecular basis of CFTR functioning. They have in particular shed light on the interdomain interfaces which are critical for signal transmission and can thus be considered for in silico screening of drugs (correctors and modulators) capable of filling the cavities displayed at these interfaces in the wild type or mutated protein. However, the modeling methodology does largely influence the characteristics of those interdomain interfaces. Here, relying on all the available experimental data on ABC transporters (full length and isolated domains), we show that there is a considerable structural plasticity in these regions which allows to adapt the partner domains. The careful consideration of such a structural plasticity in our modeling strategy leads us to propose at present four models of human CFTR, two (based on the Sav1866 and MsbA structures) in a similar open channel conformation and two (based on the MsbA and P-gp structures) in notably different closed channel conformations. We interestingly show that the global features of the transmission interfaces [between the membrane-spanning domains (MSDs) and the nucleotide binding domains (NBDs)] are remarkably conserved in these models between the open and closed forms. Further investigation of the details of the MSDs/NBDs interface also suggests that subtle differences may participate in the general mechanisms by which the signal is transmitted between MSDs and NBDs. Supported by the association Vaincre La Mucoviscidose, Paris, France.

49* Probing the oligomeric state of CFTR

Models of the 3D structure of full-length human CFTR described during the last years have provided new insights into the molecular basis of CFTR functioning. They have in particular shed light on the interdomain interfaces which are critical for signal transmission and can thus be considered for in silico screening of drugs (correctors and modulators) capable of filling the cavities displayed at these interfaces in the wild type or mutated protein. However, the modeling methodology does largely influence the characteristics of those interdomain interfaces. Here, relying on all the available experimental data on ABC transporters (full length and isolated domains), we show that there is a considerable structural plasticity in these regions which allows to adapt the partner domains. The careful consideration of such a structural plasticity in our modeling strategy leads us to propose at present four models of human CFTR, two (based on the Sav1866 and MsbA structures) in a similar open channel conformation and two (based on the MsbA and P-gp structures) in notably different closed channel conformations. We interestingly show that the global features of the transmission interfaces [between the membrane-spanning domains (MSDs) and the nucleotide binding domains (NBDs)] are remarkably conserved in these models between the open and closed forms. Further investigation of the details of the MSDs/NBDs interface also suggests that subtle differences may participate in the general mechanisms by which the signal is transmitted between MSDs and NBDs. Supported by the association Vaincre La Mucoviscidose, Paris, France.