Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA.
Department of Cell Biology and Biochemistry, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA.
Biochim Biophys Acta Biomembr. 2018 May;1860(5):1193-1204. doi: 10.1016/j.bbamem.2018.02.006. Epub 2018 Feb 7.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric T > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.
囊性纤维化跨膜电导调节因子（CFTR）是一种 ABC 转运蛋白，包含两个跨膜结构域，形成氯离子通道，以及两个核苷酸结合结构域（NBD1 和 NBD2）。CFTR 一直是获得单分散、生物物理稳定蛋白的巨大挑战。在这里，我们报告了一项综合研究，比较了单个和多个 NBD1 突变对 NBD1 结构域及其纯化的全长人 CFTR 的稳定性的影响。单个突变 S492P、A534P、I539T 表现出累加效应，当与 M470V、S495P 和 R555K 组合时，NBD1 的结构稳定性得到了显著提高。这些突变的策略性组合强烈稳定了该结构域，使其达到了热容量 T>70°C。对最稳定的 6SS-NBD1 变体进行的复制交换分子动力学模拟表明，波动、静电相互作用和侧链堆积可能是稳定性提高的原因。NBD1 的逐步稳定与全长 CFTR 蛋白结构稳定性的增强直接相关。通过内在荧光变化监测稳定 CFTR 突变体的热解折叠表明，6SS-CFTR 的 Tm 可以高达 67.4°C，比野生型高 20°C 以上。NBD2 突变 H1402S 赋予 CFTR 额外的热稳定性，可能通过稳定 NBD 二聚化构象来实现。具有 NBD1 稳定突变的 CFTR 变体在哺乳动物细胞表面表达，表现出 ATP 酶和通道活性，并在更高温度下保留这些功能。能够产生具有改善的结构稳定性的酶活性 CFTR，使其适合生物物理和结构研究，将推进机制研究和未来的囊性纤维化药物开发。
