Jamshad Mohammed, Charlton Jack, Lin Yu-Pin, Routledge Sarah J, Bawa Zharain, Knowles Timothy J, Overduin Michael, Dekker Niek, Dafforn Tim R, Bill Roslyn M, Poyner David R, Wheatley Mark
*School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
†School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K.
Biosci Rep. 2015 Apr 16;35(2):e00188. doi: 10.1042/BSR20140171.
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
G蛋白偶联受体(GPCRs)是最大的一类膜蛋白,也是主要的药物靶点。研究GPCR结构/功能特性的一个严重障碍是需要将受体从其在质膜中的天然环境中提取出来,同时GPCRs在其溶解所需的去污剂中具有固有的不稳定性。在本研究中,我们报告了首次在任何阶段完全不存在去污剂的情况下,通过利用苯乙烯马来酸(SMA)共聚物直接从膜自发包封到纳米级SMA脂质颗粒(SMALP)中,实现了功能性GPCR[人腺苷A2A受体(A2AR)]的溶解和纯化。此外,与用去污剂[DDM(正十二烷基-β-D-麦芽糖苷)]溶解的A2AR对照相比,由酵母(毕赤酵母)或哺乳动物细胞产生的A2AR-SMALP表现出更高的热稳定性(约5°C)。A2AR-SMALP在4°C下长期储存时也很稳定,并且耐多次冻融循环,这与去污剂溶解的受体形成鲜明对比。这些特性确立了在基于受体的药物发现测定中使用GPCR-SMALP的潜力。此外,与由支架蛋白稳定的纳米盘不同,SMA聚合物的非蛋白质性质使得能够对嵌入的受体进行清晰的生物物理表征。因此,利用圆二色光谱(CD光谱)将二级结构的变化与配体结合([(3)H]ZM241385)能力的丧失联系起来。GPCRs的SMALP溶解保留了环形脂质环境,将能够以类似天然的状态制备广泛的治疗靶点,以帮助药物发现和理解GPCR分子机制。