CFTR: New insights into structure and function and implications for modulation by small molecules

Kleizen, Bertrand; Hunt, J.F.; Callebaut, Isabelle; Hwang, Tzyh Chang; Sermet‐Gaudelus, Isabelle; Hafkemeyer, Sylvia; Sheppard, David N.

doi:10.1016/j.jcf.2019.10.021

Cited by 18 publications

(10 citation statements)

References 56 publications

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“…If so, the binding site must contain other residues as well, because our K166 mutants (to E or Q) showed a strong response rather than the lack of response expected upon disruption of the drug binding site. To determine the VX-809 binding site, we may need to await high-resolution structures of CFTR complexed with the drug (reviewed in [80]).…”

Section: How Where and When Does Vx-809 Act: Site-of-binding Vs Sitmentioning

confidence: 99%

Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

Kleizen

Willigen

Mijnders

et al. 2020

Preprint

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ABC-transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography, with regulation driven by the cytosolic domains, and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the protein defective in the most abundant rare inherited disease cystic fibrosis, by combining biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies a different mode of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

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Section: How Where and When Does Vx-809 Act: Site-of-binding Vs Sitmentioning

confidence: 99%

Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

Kleizen

Willigen

Mijnders

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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“…We therefore speculated that ICL2 coupling to NBD2 may be crucial for CFTR activity. Comparing solvent accessibility of K273 in Cryo-EM models of inactive CFTR 9 (PDB 5UAK) and activated, dimerized CFTR 14 (PDB 5UAR) revealed that K273 was solvent excluded in both models as well as in additional homology models 15 .…”

Section: Resultsmentioning

confidence: 99%

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

Pankow

Bamberger

Martínez‐Bartolomé

et al. 2021

Preprint

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In vivo characterization of protein structures or protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein complexes. Using a novel low-resolution method named Covalent Protein Painting (CPP) that allows the characterization of protein conformations in vivo, we are the first to report how an ion channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), is conformationally changed during biogenesis and channel opening in the cell. Our study led to the identification of a novel opening mechanism for CFTR by revealing that the interaction of the intracellular loop 2 (ICL2) with the nucleotide binding domain 2 (NDB2) of CFTR is needed for channel gating, and this interaction occurs concomitantly with changes to the narrow part of the pore and the walker A lysine in NBD1. However, the ICL2:NBD2 interface, which forms a "ball-in-a-socket" motif, is uncoupled during biogenesis, likely to prevent inadvertent channel activation during transport. In particular, solvent accessibility of lysine 273 (K273) in ICL2 changes with the opening and closing of the channel. Mutation of K273 severely impaired CFTR biogenesis and led to accumulation of CFTR in the Golgi and TGN. CPP further revealed that, even upon treatment with current approved drugs or at permissive temperature, the uncoupled state of ICL2 is a prominent feature of the misfolded CFTR mutants ΔF508 and N1303K that cause Cystic Fibrosis (CF), which suggests that stabilization of this interface could produce a more efficient CF drug. CPP is able to characterize a protein in its native environment and measure the effect of complex PTMs and protein interactions on protein structure, making it broadly applicable and invaluable for the development of new therapies.

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“…Experts in the CF field still maintain that "structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about CFTR and are key for the development of better drugs to enable people with CF to live full and active lives" [44,45]. In addition, scientists are comparing and trying to elucidate the robustness of current methods and markers used to evaluate the benefit of these new modulation therapies [27,46,47].…”

Section: Patient Response To Cftr Modulatorsmentioning

confidence: 99%

The era of CFTR modulators: improvements made and remaining challenges

Cuevas-Ocaña

Laselva

Avolio

et al. 2020

Breathe

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Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene [1]. The CFTR protein is an ion channel that mediates chloride and bicarbonate transport in epithelial cells of multiple organs including lungs, pancreas and intestine [2, 3]. A defective CFTR protein produces an impaired ion and fluid secretion in the epithelial cells affecting several organs and leading to severe lung disease. More than 2000 CF-causing mutations have been identified [4, 5]. The most common mutation, the deletion of phenylalanine at position 508 (F508del), induces misfolding of the protein that is retained in the endoplasmic reticulum and degraded by proteasomal pathways [6].

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CFTR: New insights into structure and function and implications for modulation by small molecules

Cited by 18 publications

References 56 publications

Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

The era of CFTR modulators: improvements made and remaining challenges

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