Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ΔF508 CFTR Folding and Assembly

JOURNAL OF MOLECULAR BIOLOGY

卷:427

Article

He, Lihua¹  Aleksandrov, Andrei A.¹  An, Jianli²  Cui, Liying¹  Yang, Zhengrong^2,3  Brouillette, Christie G.^2,3  Riordan, John R.¹ 

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

[2] Univ Alabama Birmingham, Struct Biol Ctr, Birmingham, AL 35294 USA

[3] Univ Alabama Birmingham, Dept Chem, Birmingham, AL 35294 USA

关键词: cystic fibrosis;    CFTR;    thermal stability;    ion channel;    protein folding;   

DOI  :  10.1016/j.jmb.2014.07.026

来源: Elsevier

【 摘 要 】

Cystic fibrosis transmennbrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (Delta F508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The Delta F508 mutation has two major impacts on CFTR: reduced thermal Stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on Delta F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the Delta F508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function. (C) 2014 Published by Elsevier Ltd.

【 授权许可】

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