Dataset Information

Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange.

ABSTRACT: Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site.

SUBMITTER: Byrnes LJ

PROVIDER: S-EPMC5856801 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange.

Byrnes Laura J LJ Xu Yingrong Y Qiu Xiayang X Hall Justin D JD West Graham M GM

Scientific reports 20180316 1

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spe ...[more]

PMID: 29549268

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange.

Publications

Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange.

Similar Datasets

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