Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.


Abstract

The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R 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 A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-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. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.

Submission Details

ID: Xvd2buam3

Submitter: Paul Chang

Submission Date: June 5, 2019, 4:07 p.m.

Version: 1

Publication Details
Lebon G;Bennett K;Jazayeri A;Tate CG,J Mol Biol (2011) Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor. PMID:21501622
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2YDO 2011-03-23T00:00:00+0000 3.0 Thermostabilised HUMAN A2a Receptor with adenosine bound
2YDV 2011-03-24T00:00:00+0000 2.6 Thermostabilised HUMAN A2a Receptor with NECA bound
3EML 2008-09-24T00:00:00+0000 2.6 The 2.6 A Crystal Structure of a Human A2A Adenosine Receptor bound to ZM241385.
3PWH 2010-12-08T00:00:00+0000 3.3 Thermostabilised Adenosine A2A Receptor
3QAK 2011-01-11T00:00:00+0000 2.71 Agonist bound structure of the human adenosine A2a receptor
3REY 2011-04-05T00:00:00+0000 3.31 Thermostabilised adenosine A2A receptor in complex with XAC
3RFM 2011-04-06T00:00:00+0000 3.6 Thermostabilised adenosine A2A receptor in complex with caffeine
3UZA 2011-12-07T00:00:00+0000 3.27 Thermostabilised Adenosine A2A receptor in complex with 6-(2,6-Dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-3-amine
3UZC 2011-12-07T00:00:00+0000 3.34 Thermostabilised Adenosine A2A receptor in complex with 4-(3-amino-5-phenyl-1,2,4-triazin-6-yl)-2-chlorophenol
3VG9 2011-08-04T00:00:00+0000 2.7 Crystal structure of human adenosine A2A receptor with an allosteric inverse-agonist antibody at 2.7 A resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Adenosine receptor A2a P29274 AA2AR_HUMAN
93.2 Adenosine receptor A2a P11617 AA2AR_CANLF