A topological switch in CFTR modulates channel activity and sensitivity to unfolding

Scholl, Daniel; Sigoillot, Maud; Overtus, Marie; Martinez, Rafael Colomer; Martens, Chloé; Wang, Yiting; Pardon, Els; Laeremans, Toon; García-Pino, Abel; Steyaert, Jan; Sheppard, David N.; Hendrix, Jelle; Govaerts, Cédric

doi:10.1038/s41589-021-00844-0

Cited by 20 publications

(18 citation statements)

References 60 publications

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“…Functionally, a dynamic ICL4/NBD1 interface is crucial for CFTR gating, as demonstrated by the rapid and reversible interruption of channel activity that occurs upon formation of covalent cross-links between F508C and F1068C in a cys-less CFTR background ( 21 ). In addition, recent studies suggest that a frequent “uncoupling” of NBD1 is an integral feature of WT CFTR gating ( 20 , 78 ). A tryptophan side chain at position R1070, capable of both hydrogen bonding and hydrophobic contacts, would allow the protein to adopt conformations in which the interface is alternatively buried or more solvent exposed.…”

Section: Discussionmentioning

confidence: 99%

“…Possibly the absence of a short helix in NBD1—present in NBD2 and in the NBDs of other ABC transporters ( 19 )—is responsible for a shallower socket and weaker ICL4/NBD1 interactions, rendering the ICL4/NBD1 interface particularly vulnerable to harmful mutations. Moreover, a more dynamic NBD1 structure around the socket might also contribute to causing this mutation hotspot ( 20 ).…”

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confidence: 99%

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Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Prins

Corradi

Sheppard

et al. 2022

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Prins

Corradi

Sheppard

et al. 2022

Journal of Biological Chemistry

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“…Our multi-domain models are still missing many loop regions that remain undetermined in the cryo-EM density. For example, the regulatory insertion (RI) region changes the thermodynamic stability of CFTR (36) and adopts distinct conformations, one of which has been postulated to lead to ΔF508 unfolding (37). Our models are also missing the R domain (a large unstructured 200 residues between NBD1 and TMD2), the glycosylation site, and the loop connecting TMD2 to NBD2.…”

Section: Discussionmentioning

confidence: 99%

Structural comparative modeling of multi-domain ΔF508 CFTR

McDonald

Woods

Smith

et al. 2021

Preprint

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Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (ΔF508), making ΔF508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain ΔF508 CFTR structure has been determined and few studies have modeled ΔF508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a ΔF508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and ΔF508 CFTR structural models. These models demonstrate the destabilizing effects of ΔF508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled ΔF508/R1070W and ΔF508 bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of ΔF508 CFTR and pave the way for rational design of additional drugs that target ΔF508 CFTR for treatment of CF.Author SummaryProtein’s three-dimension shape determines their function, so when genetic mutation compromises the shape of vital proteins, it may cause disease. Such is the case in Cystic Fibrosis, a chronic genetic disease caused by mutations in the protein Cystic Fibrosis Transmembrane Conductance Regulator. Here, we work backwards from the shape of the wild-type protein – found in healthy people, to computationally model the shape of the most common Cystic Fibrosis mutant. Our computer models reveal distinct defects in the shape of the mutant Cystic Fibrosis Transmembrane Conductance Regulator protein in the area surrounding the mutation. We also model an important FDA approved Cystic Fibrosis drug, VX-809, into the mutant protein structure and show how VX-809 stabilizes the protein around the location of the mutation. The method we developed will pave the way for computational drug design for Cystic Fibrosis.

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“…Functionally, a dynamic ICL4/NBD1 interface is crucial for CFTR gating, as demonstrated by the rapid and reversible interruption of channel activity that occurs upon formation of covalent cross-links between F508C and F1068C in a cys-less CFTR background (21). In addition, recent studies suggest that a frequent "uncoupling" of NBD1 is an integral feature of WT CFTR gating (20,77). A tryptophan side-chain at position R1070, capable of both hydrogen bonding and hydrophobic contacts, would allow the protein to adopt conformations in which the interface is alternatively buried or more solvent exposed.…”

Section: F508del/r1070w Restores Icl4/nbd1 Hydrogen-bond Interactions Allowing Flexibility Of the Nbd1 Loopmentioning

confidence: 99%

“…Possibly the absence of a short helix in NBD1present in NBD2 and in the NBDs of other ABC transporters (19) is responsible for a shallower socket and weaker ICL4/NBD1 interactions, rendering the ICL4/NBD1 interface particularly vulnerable to harmful mutations. Moreover, a more dynamic NBD1 structure around the socket might also contribute to causing this mutation hotspot (20).…”

Section: Introductionmentioning

confidence: 99%

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Prins

Corradi

Sheppard

et al. 2021

Preprint

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Deletion of phenylalanine 508 (F508del), in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, is the most common cause of cystic fibrosis (CF). F508 is located on nucleotide-binding domain 1 (NBD1) in contact with cytosolic extensions of transmembrane helices, in particular intracellular loop 4 (ICL4). We carried out a mutagenesis scan of ICL4 by introducing five or six second-site mutations at eleven positions in cis with F508del, and quantifying changes in membrane proximity and ion-channel function of CFTR. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains, (F1068, R1070W, and F1074, mimicking F1068, F508 and F1074 in wild-type CFTR). F508del-CFTR had a distorted aromatic stack, with F1068 displaced towards space vacated by F508. In F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds, and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR are F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. Finally, two mutations identified in a yeast scan (A141S and R1097T, on adjacent transmembrane helices linked to ICL1 and ICL4) also partially rescued F508del-CFTR function. These studies highlight the importance of hydrophobic interactions and conformational flexibility at the ICL4/NBD1 interface, advancing understanding of the structural underpinning of F508del dysfunction.

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A topological switch in CFTR modulates channel activity and sensitivity to unfolding

Cited by 20 publications

References 60 publications

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Structural comparative modeling of multi-domain ΔF508 CFTR

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

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