Regulatory Insertion Removal Restores Maturation, Stability and Function of ΔF508 CFTR

JOURNAL OF MOLECULAR BIOLOGY

卷:401

Article

Aleksandrov, Andrei A.^1,2  Kota, Pradeep^3,4  Aleksandrov, Luba A.^1,3  He, Lihua^1,3  Jensen, Tim^1,3  Cui, Liying^1,3  Gentzsch, Martina^1,5  Dokholyan, Nikolay V.^3,4  Riordan, John R.^1,3 

[1] Univ N Carolina, Cyst Fibrosis Treatment & Res Ctr, Chapel Hill, NC 27599 USA

[2] Univ N Carolina, Dept Biomed Engn, Chapel Hill, NC 27599 USA

[3] Univ N Carolina, Dept Biochem & Biophys, Chapel Hill, NC 27599 USA

[4] Univ N Carolina, Mol & Cellular Biophys Program, Chapel Hill, NC 27599 USA

[5] Univ N Carolina, Dept Cell & Dev Biol, Chapel Hill, NC 27599 USA

关键词: ABC transporters;    CFTR;    cystic fibrosis;    ion channel;    DMD simulations;   

DOI  :  10.1016/j.jmb.2010.06.019

来源: Elsevier

【 摘 要 】

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 Delta RI/Delta F508 had a T-1/2 of similar to 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of Delta F508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees 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. (C) 2010 Elsevier Ltd. All rights reserved.

【 授权许可】

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