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Biochim Biophys Acta Biomembr. 2018 May;1860(5):1193-1204. doi: 10.1016/j.bbamem.2018.02.006. Epub 2018 Feb 7.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

Author information

1
Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA.
2
Department of Cell Biology and Biochemistry, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA.
3
Department of Medicine, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA.
4
Department of Biochemistry and Biophysics and Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
5
Department of Chemistry, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
6
Department of Medicine, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA; Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233, USA.
7
Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: christie@uab.edu.
8
Department of Cell Biology and Biochemistry, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA. Electronic address: ina.urbatsch@ttuhsc.edu.

Abstract

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

KEYWORDS:

ABC transporters; ATP hydrolysis; CFTR; NBD1; Protein unfolding; Stabilizing mutations; Thermal stability

PMID:
29425673
PMCID:
PMC6319260
DOI:
10.1016/j.bbamem.2018.02.006
[Indexed for MEDLINE]
Free PMC Article

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