Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR

Andrei A. Aleksandrov, Pradeep Kota, Luba A. Aleksandrov, Lihua He, Tim Jensen, Liying Cui, Martina Gentzsch, Nikolay Dokholyan, John R. Riordan

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

Original languageEnglish (US)
Pages (from-to)194-210
Number of pages17
JournalJournal of Molecular Biology
Volume401
Issue number2
DOIs
StatePublished - Aug 1 2010

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Cystic Fibrosis Transmembrane Conductance Regulator
Anions
Adenosine Triphosphate
Chloride Channels
ATP-Binding Cassette Transporters
Molecular Dynamics Simulation
Cystic Fibrosis
Quality Control
Membrane Proteins
Nucleotides
Peptides
Mutation
Proteins

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Cite this

Aleksandrov, A. A., Kota, P., Aleksandrov, L. A., He, L., Jensen, T., Cui, L., ... Riordan, J. R. (2010). Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR. Journal of Molecular Biology, 401(2), 194-210. https://doi.org/10.1016/j.jmb.2010.06.019
Aleksandrov, Andrei A. ; Kota, Pradeep ; Aleksandrov, Luba A. ; He, Lihua ; Jensen, Tim ; Cui, Liying ; Gentzsch, Martina ; Dokholyan, Nikolay ; Riordan, John R. / Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR. In: Journal of Molecular Biology. 2010 ; Vol. 401, No. 2. pp. 194-210.
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abstract = "The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.",
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Aleksandrov, AA, Kota, P, Aleksandrov, LA, He, L, Jensen, T, Cui, L, Gentzsch, M, Dokholyan, N & Riordan, JR 2010, 'Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR', Journal of Molecular Biology, vol. 401, no. 2, pp. 194-210. https://doi.org/10.1016/j.jmb.2010.06.019

Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR. / Aleksandrov, Andrei A.; Kota, Pradeep; Aleksandrov, Luba A.; He, Lihua; Jensen, Tim; Cui, Liying; Gentzsch, Martina; Dokholyan, Nikolay; Riordan, John R.

In: Journal of Molecular Biology, Vol. 401, No. 2, 01.08.2010, p. 194-210.

Research output: Contribution to journalArticle

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T1 - Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR

AU - Aleksandrov, Andrei A.

AU - Kota, Pradeep

AU - Aleksandrov, Luba A.

AU - He, Lihua

AU - Jensen, Tim

AU - Cui, Liying

AU - Gentzsch, Martina

AU - Dokholyan, Nikolay

AU - Riordan, John R.

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N2 - The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

AB - The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

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