Allosteric Coupling between the Intracellular Coupling Helix 4 and Regulatory Sites of the First Nucleotide-binding Domain of CFTR

Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.

囊性纤维化（Cystic fibrosis）由CFTR（囊性纤维化跨膜传导调节因子，cystic fibrosis transmembrane conductance regulator）的突变引发，可导致蛋白质折叠与加工缺陷，以及氯离子通道门控功能异常。CFTR的调控依赖于ATP结合至其胞质核苷酸结合结构域（nucleotide-binding domains, NBD1和NBD2），以及NBD1的调控插入区（regulatory insert, RI）与调控延伸区（regulatory extension, RE）/R区域的磷酸化。这些调控效应通过NBD与细胞内结构域偶联螺旋（intracellular domain coupling helices, CL）——尤其是CL4——之间的相互作用，传递至通道的其余区域。利用一种可检测核磁共振（Nuclear Magnetic Resonance, NMR）光谱中化学位移变化对应的残基间相关性的灵敏方法，研究人员在缺失RI的NBD1构建体内部揭示了一条变构网络。CL4结合位点与RI缺失位点，以及全长CFTR中R区域上游的NBD1 C端残基形成偶联。向NBD1中滴定CL4肽段会扰动这些位点的构象集合，且在主要囊性纤维化致病突变体F508del、抑制突变体F494N、V510D和Q637R NBD1，以及CL4-NBD1融合构建体中均观察到了相似的滴定模式。反之，C端突变Q637R会扰动这三个位点的动力学特性。该变构网络揭示了一种整合多种NBD1调控相互作用的机制，同时为CFTR功能所必需的变构偶联提供了生物物理证据。