CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor

Georg Krainer,Mathias Schenkel,Andreas Hartmann,Dorna Ravamehr-Lake,Charles M Deber,Michael Schlierf

doi:10.1074/jbc.ac119.011360

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel protein that is defective in individuals with cystic fibrosis (CF). To advance the rational design of CF therapies, it is important to elucidate how mutational defects in CFTR lead to its impairment and how pharmacological compounds interact with and alter CFTR. Here, using a helical-hairpin construct derived from CFTR's transmembrane (TM) helices 3 and 4 (TM3/4) and their intervening loop, we investigated the structural effects of a patient-derived CF-phenotypic mutation, E217G, located in the loop region of CFTR's membrane-spanning domain. Employing a single-molecule FRET assay to probe the folding status of reconstituted hairpins in lipid bilayers, we found that the E217G hairpin exhibits an altered adaptive packing behavior stemming from an additional GXXXG helix-helix interaction motif created in the mutant hairpin. This observation suggested that the misfolding and functional defects caused by the E217G mutation arise from an impaired conformational adaptability of TM helical segments in CFTR. The addition of the small-molecule corrector Lumacaftor exerts a helix stabilization effect not only on the E217G mutant hairpin, but also on WT TM3/4 and other mutations in the hairpin. This finding suggests a general mode of action for Lumacaftor through which this corrector efficiently improves maturation of various CFTR mutants.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel protein that is defective in individuals with cystic fibrosis (CF)
Employing a single-molecule FRET assay to probe the folding status of reconstituted hairpins in lipid bilayers, we found that the E217G hairpin exhibits an altered adaptive packing behavior stemming from an additional GXXXG helix–helix interaction motif created in the mutant hairpin
Among the two CFTR mutations found in the extracellular loop region connecting TM3 and TM helix 4 (TM4), we focus here on the disease-causing loop mutation E217G (Fig. 1a), which causes a mild form of the disease and drastically alters net charge and hydrophobicity of the loop region

Summary

Edited by Ursula Jakob

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel protein that is defective in individuals with cystic fibrosis (CF). Employing a single-molecule FRET assay to probe the folding status of reconstituted hairpins in lipid bilayers, we found that the E217G hairpin exhibits an altered adaptive packing behavior stemming from an additional GXXXG helix–helix interaction motif created in the mutant hairpin This observation suggested that the misfolding and functional defects caused by the E217G mutation arise from an impaired conformational adaptability of TM helical segments in CFTR. CFTR with its 1,480 amino acid residues is too large and too complex to pinpoint the local structural effects of a single point mutation, for classical ensemble biochemical and biophysical techniques, which are often limited in their ability to resolve the structural heterogeneities of misfolded states To overcome these difficulties, we recently introduced a single-molecule approach that exploits helical-hairpin constructs derived from full-length CFTR to gain insights into the structural effects of misfolding and drug rescue [13]. We and others have previously shown that Lumacaftor efficiently targets the first membrane-spanning domain of CFTR, including the TM3/4 hairpin, to rescue misfolding of mutations located in TM helices [13, 29,30,31,32,33], yet the potential effects of Lumacaftor on misfolding mutations located in loop regions, such as E217G, are unexplored

Results and discussion

Conclusion

Experimental procedures