Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure

Hal A. Lewis, Xun Zhao, Chi Wang, J. Michael Sauder, Isabelle Rooney, Brian W. Noland, Don Lorimer, Margaret C. Kearins, Kris Conners, Brad Condon, Peter C. Maloney, William B Guggino, John F. Hunt, Spencer Emtage

Research output: Contribution to journalArticle

Abstract

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (ΔF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ΔF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ΔF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ΔF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ΔF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

Original languageEnglish (US)
Pages (from-to)1346-1353
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number2
DOIs
StatePublished - Jan 14 2005
Externally publishedYes

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Cystic Fibrosis Transmembrane Conductance Regulator
Nucleotides
Mutation
Conformations
Genetic Suppression
Defects
Protein Conformation
Proteins
Surface topography
Cystic Fibrosis
Adenosine Triphosphatases
Catalytic Domain
Crystal structure
Epithelial Cells
Membranes
Kinetics
Population

ASJC Scopus subject areas

  • Biochemistry

Cite this

Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. / Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B; Hunt, John F.; Emtage, Spencer.

In: Journal of Biological Chemistry, Vol. 280, No. 2, 14.01.2005, p. 1346-1353.

Research output: Contribution to journalArticle

Lewis, HA, Zhao, X, Wang, C, Sauder, JM, Rooney, I, Noland, BW, Lorimer, D, Kearins, MC, Conners, K, Condon, B, Maloney, PC, Guggino, WB, Hunt, JF & Emtage, S 2005, 'Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure', Journal of Biological Chemistry, vol. 280, no. 2, pp. 1346-1353. https://doi.org/10.1074/jbc.M410968200
Lewis, Hal A. ; Zhao, Xun ; Wang, Chi ; Sauder, J. Michael ; Rooney, Isabelle ; Noland, Brian W. ; Lorimer, Don ; Kearins, Margaret C. ; Conners, Kris ; Condon, Brad ; Maloney, Peter C. ; Guggino, William B ; Hunt, John F. ; Emtage, Spencer. / Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 2. pp. 1346-1353.
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AU - Zhao, Xun

AU - Wang, Chi

AU - Sauder, J. Michael

AU - Rooney, Isabelle

AU - Noland, Brian W.

AU - Lorimer, Don

AU - Kearins, Margaret C.

AU - Conners, Kris

AU - Condon, Brad

AU - Maloney, Peter C.

AU - Guggino, William B

AU - Hunt, John F.

AU - Emtage, Spencer

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N2 - Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (ΔF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ΔF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ΔF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ΔF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ΔF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

AB - Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (ΔF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ΔF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ΔF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ΔF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ΔF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

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