Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure
Impact of the ΔF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure
复制标题
DOI：
10.1074/jbc.m410968200
复制
发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Emtage, S
中科院分区：
文献类型：
--
作者：
Lewis, HA;Zhao, X;Emtage, S
囊性纤维化是由囊性纤维化跨膜传导调节因子(CFTR)的缺陷引起的，通常是由于第一核苷酸结合域(NBD1)中Phe-508(DeltaF508)残基的缺失，导致上皮细胞表面功能通道的数量严重减少。以前使用不完全NBD1结构域的研究将其归因于DeltaF508 NBD1的异常折叠。我们报告了对完整的人类NBD1结构域的结构和生物物理研究，这些研究未能证明在存在或不存在DeltaF508突变的情况下体外稳定性或折叠动力学的显著变化。晶体结构显示，蛋白质构象变化很小，但突变部位的局部表面形貌发生了显著变化，该突变部位位于NBD1区域，据信与CFTR的第一个跨膜结构域相互作用。这些结果提出了这样一种可能性，即DeltaF508的主要作用是破坏CFTR中该位点的正确结构域间相互作用，而不是干扰NBD1的折叠。有趣的是，在引入抑制DeltaF508突变引起的运输缺陷的第二位点突变后，观察到NBD1结构的稳定性增加，这表明这些抑制物可能通过提高NBD1在全长蛋白质中的折叠效率来间接发挥作用。人类NBD1的结构也巩固了对CFTR调控的理解，因为它的两个可磷酸化的蛋白质片段都采用了多种构象，调节对ATPase活性位点和功能域间界面的访问。
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator ( CFTR), commonly the deletion of residue Phe-508 (DeltaF508) 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 DeltaF508 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 DeltaF508 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 DeltaF508 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 DeltaF508 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.