Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini, 6, 16149 Genoa, Italy.
Cell Mol Life Sci. 2012 Nov;69(21):3701-13. doi: 10.1007/s00018-012-1049-7. Epub 2012 Jul 3.
Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs-intracellular loop interaction in a way that would facilitate the open state of the channel.
核苷酸结合域（NBD1 和 NBD2）的囊性纤维化跨膜电导调节因子（CFTR），在囊性纤维化中的缺陷蛋白，负责控制氯离子通道的门控，并且是疾病治疗中几种候选药物的潜在结合位点。我们研究了应用 2-嘧啶-7,8-苯并黄酮（PBF），CFTR 的一种强增敏剂，对重组和等摩尔 NBD1/NBD2 混合物在溶液中的性质的影响。结果表明，增敏剂诱导 NBD1/NBD2 二聚体在溶液中发生显著的构象变化。增敏剂不改变 ATP 结合常数，但降低 NBD1/NBD2 混合物的 ATP 水解活性。本征荧光和胍变性测量表明，增敏剂在存在和不存在 ATP 的情况下，对 NBD1/NBD2 混合物产生不同的构象变化。从小角 X 射线散射实验证实，在没有 ATP 的情况下，NBD1/NBD2 二聚体被增敏剂破坏，但在存在 2 mM ATP 的情况下，两个 NBD 保持二聚化，并观察到结构的大小和形状发生了重大变化。我们提出，这些构象变化可以改变 NBDs-细胞内环相互作用的方式，从而促进通道的开放状态。
