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J Biol Chem. 2017 Aug 25;292(34):14147-14164. doi: 10.1074/jbc.M116.772335. Epub 2017 Jun 27.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Author information

1
From the Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
2
the Center for Structural Biology and Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294.
3
the Department of Biochemistry and Biophysics, Cystic Fibrosis Treatment and Research Center, University of North Carolina, Chapel Hill, North Carolina 27599, and.
4
the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219.
5
From the Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada, forman@sickkids.ca.
6
the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Abstract

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF.

KEYWORDS:

ABC transporter; cystic fibrosis transmembrane conductance regulator (CFTR); protein engineering; protein misfolding; protein stability

PMID:
28655774
PMCID:
PMC5572908
DOI:
10.1074/jbc.M116.772335
[Indexed for MEDLINE]
Free PMC Article

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