Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor
Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor
复制标题
DOI：
10.1073/pnas.0804396105
复制
发表时间：
2008-08-05
影响因子：
11.1
通讯作者：
Tate, Christopher G.
中科院分区：
文献类型：
--
作者：
Magnani, Francesca;Shibata, Yoko;Tate, Christopher G.
由于哺乳动物整体膜蛋白在洗涤剂中的不稳定性，其结构研究一直受到阻碍。G蛋白偶联受体（gpcr）的激动剂构象尤其如此，人们认为，在激动剂结合时发生的螺旋运动导致蛋白质中更松散和更不稳定的包装。在这里，我们表明，诱变与特定的选择策略相结合，可以用来稳定腺苷a （2a）受体的激动剂和拮抗剂构象。在鉴定的27个改善激动剂构象热稳定性的突变中，在鉴定的17个改善拮抗剂构象热稳定性的突变中，只有3个也存在，这表明所使用的选择策略是针对每种构象的。拮抗剂或激动剂结合构象的稳定突变组合导致突变体在高温下比野生型受体分别稳定17℃和9℃。与野生型受体相比，突变体受体在短链烷基糖苷洗涤剂中的稳定性都有显著提高，这将有助于它们的结构分析。
Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A(2a) receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist- or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17 degrees C and 9 degrees C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.