CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor
CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor
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DOI：
10.1074/jbc.ac119.011360
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发表时间：
2020-02-14
影响因子：
4.8
通讯作者：
Schlierf, Michael
中科院分区：
文献类型：
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作者：
Krainer, Georg;Schenkel, Mathias;Schlierf, Michael
囊性纤维化跨膜电导调节蛋白(CFTR)是一种离子通道蛋白，在囊性纤维化患者中存在缺陷。为了促进CF治疗的合理设计，阐明CFTR的突变缺陷是如何导致其损伤的，以及药物如何与CFTR相互作用和改变CFTR是很重要的。在这里，使用来自CFTR的跨膜(TM)螺旋3和4(TM3/4)及其干预环的螺旋发夹结构，我们研究了患者来源的CF表型突变E217G的结构效应，该突变位于CFTR跨膜结构域的环区。利用单分子FRET实验探索重组发夹在脂质双层中的折叠状态，我们发现E217G发夹表现出一种改变的适应性堆积行为，这源于突变发夹中额外的GXXXG螺旋相互作用基序。这一观察结果表明，E217G突变导致的错误折叠和功能缺陷是由于CFTR中TM螺旋片段的构象适应性受损所致。小分子校正剂Lumacaftor的加入不仅对E217G突变发夹起到螺旋稳定作用，还对WT TM3/4等发夹突变起到稳定作用。这一发现提示了Lumacaftor的一般作用模式，通过该模式，该校正器有效地促进了各种CFTR突变体的成熟。
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.