Cui Tanxing, Mowrey David, Bondarenko Vasyl, Tillman Tommy, Ma Dejian, Landrum Elizabeth, Perez-Aguilar Jose Manuel, He Jing, Wang Wei, Saven Jeffery G, Eckenhoff Roderic G, Tang Pei, Xu Yan
Department of Anesthesiology, University of Pittsburgh School of Medicine, PA, USA.
Biochim Biophys Acta. 2012 Mar;1818(3):617-26. doi: 10.1016/j.bbamem.2011.11.021. Epub 2011 Dec 3.
The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.
烟碱型乙酰胆碱受体（nAChR）是多种病理生理状况下重要的治疗靶点，针对该靶点进行合理的药物设计通常需要原子分辨率的受体结构。最近的概念验证研究展示了一种通过核磁共振（NMR）确定膜蛋白结构的水溶性方法（斯洛维奇等人，《美国国家科学院院刊》，101: 1828 - 1833, 2004；马等人，《美国国家科学院院刊》，105: 16537 - 16542, 2008）。我们在此报告WSA的计算设计和实验表征，WSA是一种水溶性蛋白，与nAChRα1亚基的跨膜（TM）结构域具有约83%的序列同一性。尽管该设计基于低分辨率结构模板，但所得的高分辨率NMR结构与细菌紫球藻五聚体配体门控离子通道（GLIC）TM结构域的最新晶体结构非常吻合，证明了该方法的稳健性和普遍适用性。NMR T(2)色散测量表明，所设计蛋白的TM2结构域具有动态性，在NMR时间尺度上发生构象交换。用异氟烷和丙泊酚光亲和标记物进行的光亲和标记在麻醉剂 - GLIC复合物晶体结构中发现的麻醉剂结合位点附近确定了一个共同的结合位点。我们的结果说明了对水溶性通道蛋白进行高分辨率NMR分析对于发现潜在药物结合位点的有用性。
