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
3EML 2008-09-24T00:00:00+0000 2.6 The 2.6 A Crystal Structure of a Human A2A Adenosine Receptor bound to ZM241385.
6AQF 2017-08-19T00:00:00+0000 2.51 Crystal structure of A2AAR-BRIL in complex with the antagonist ZM241385 produced from Pichia pastoris
5NLX 2017-04-05T00:00:00+0000 2.14 A2A Adenosine receptor room-temperature structure determined by serial millisecond crystallography
2YDO 2011-03-23T00:00:00+0000 3.0 Thermostabilised HUMAN A2a Receptor with adenosine bound
5K2C 2016-05-18T00:00:00+0000 1.9 1.9 angstrom A2a adenosine receptor structure with sulfur SAD phasing and phase extension using XFEL data
5K2A 2016-05-18T00:00:00+0000 2.5 2.5 angstrom A2a adenosine receptor structure with sulfur SAD phasing using XFEL data
5K2B 2016-05-18T00:00:00+0000 2.5 2.5 angstrom A2a adenosine receptor structure with MR phasing using XFEL data
2YDV 2011-03-24T00:00:00+0000 2.6 Thermostabilised HUMAN A2a Receptor with NECA bound
5UIG 2017-01-13T00:00:00+0000 3.5 Crystal structure of adenosine A2A receptor bound to a novel triazole-carboximidamide antagonist
4EIY 2012-04-06T00:00:00+0000 1.8 Crystal structure of the chimeric protein of A2aAR-BRIL in complex with ZM241385 at 1.8A resolution

Relevant UniProtKB Entries

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