Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.
Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.
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DOI：
10.1016/j.jmb.2011.03.075
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发表时间：
2011-06-10
影响因子：
5.6
通讯作者：
Tate CG
中科院分区：
文献类型：
--
作者：
Lebon G;Bennett K;Jazayeri A;Tate CG
腺苷A2 A受体（A2 AR）是一种G蛋白偶联受体，在激动剂腺苷介导的跨膜信号传导中起关键作用。A2 AR的结构确定最近在拮抗剂结合的构象，这是促进T4溶菌酶融合在细胞质环3和相当大的稳定性赋予受体的结合反向激动剂ZM 241385。不幸的是，天然激动剂腺苷不能充分稳定受体以形成衍射质量的晶体。作为确定与激动剂结合的A2 AR结构的第一步，在结合的激动剂[3 H] 5 '-N-乙基羧酰胺腺苷（NECA）存在下，通过系统诱变使受体热稳定。鉴定了四种热稳定突变，当组合得到突变体A2 AR-GL 26时，与野生型受体相比，其解折叠速率降低了200倍以上。药理学分析表明，A2 AR-GL 26在激动剂结合构象中稳定，因为拮抗剂以高达320倍降低的亲和力结合。没有一个热稳定突变在ZM 241385结合口袋中，这表明突变通过改变受体的构象而不是通过与配体的直接相互作用来影响配体结合。A2 AR-GL 26在短链洗涤剂中显示出相当大的稳定性，这使得其能够纯化和结晶。
The adenosine A2A receptor (A2AR) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A2AR was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A2AR bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [3H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A2AR-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A2AR-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A2AR-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.
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DOI：
10.1038/nrd2760
发表时间：
2009-01
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.8
作者：
Gether, U;Ballesteros, JA;Kobilka, BK
通讯作者：
Kobilka, BK
DOI：
10.1073/pnas.0804396105
发表时间：
2008-08-05
影响因子：
11.1
作者：
Magnani, Francesca;Shibata, Yoko;Tate, Christopher G.
通讯作者：
Tate, Christopher G.
影响因子：
64.8
作者：
Rasmussen, Soren G. F.;Choi, Hee-Jung;Fung, Juan Jose;Pardon, Els;Casarosa, Paola;Chae, Pil Seok;DeVree, Brian T.;Rosenbaum, Daniel M.;Thian, Foon Sun;Kobilka, Tong Sun;Schnapp, Andreas;Konetzki, Ingo;Sunahara, Roger K.;Gellman, Samuel H.;Pautsch, Alexander;Steyaert, Jan;Weis, William I.;Kobilka, Brian K.
通讯作者：
Kobilka, Brian K.
DOI：
10.1126/science.1164772
发表时间：
2008-11-21
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Jaakola VP;Griffith MT;Hanson MA;Cherezov V;Chien EY;Lane JR;Ijzerman AP;Stevens RC
通讯作者：
Stevens RC