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
5NM4 2017-09-27 1.7 A2A Adenosine receptor room-temperature structure determined by serial femtosecond crystallography
5IU4 2016-06-29 1.72 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with ZM241385 at 1.7A resolution
4EIY 2012-07-25 1.8 Crystal structure of the chimeric protein of A2aAR-BRIL in complex with ZM241385 at 1.8A resolution
6PS7 2019-11-13 1.85 XFEL A2aR structure by ligand exchange from LUF5843 to ZM241385.
5OLG 2018-01-17 1.87 Structure of the A2A-StaR2-bRIL562-ZM241385 complex at 1.86A obtained from in meso soaking experiments.
5IU7 2016-06-29 1.9 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with compound 12c at 1.9A resolution
5K2C 2016-09-21 1.9 1.9 angstrom A2a adenosine receptor structure with sulfur SAD phasing and phase extension using XFEL data
5OLZ 2018-01-17 1.9 Structure of the A2A-StaR2-bRIL562-Compound 4e complex at 1.9A obtained from bespoke co-crystallisation experiments.
5K2D 2016-09-21 1.9 1.9A angstrom A2a adenosine receptor structure with MR phasing using XFEL data
6ZDR 2020-09-16 1.92 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with Chromone 4d
5NM2 2017-09-27 1.95 A2A Adenosine receptor cryo structure
5IU8 2016-06-29 2.0 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with compound 12f at 2.0A resolution
5OLV 2018-01-17 2.0 Structure of the A2A-StaR2-bRIL562-LUAA47070 complex at 2.0A obtained from in meso soaking experiments.
5MZJ 2017-07-26 2.0 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with theophylline at 2.0A resolution
6GT3 2019-06-26 2.0 Crystal Structure of the A2A-StaR2-bRIL562 in complex with AZD4635 at 2.0A resolution
5OM4 2018-01-17 2.0 Structure of the A2A-StaR2-bRIL562-Compound 4e complex at 1.86A obtained from in meso soaking experiments (24 hour soak).
5OM1 2018-01-17 2.1 Structure of the A2A-StaR2-bRIL562-Compound 4e complex at 2.1A obtained from in meso soaking experiments (1 hour soak).
5MZP 2017-07-26 2.1 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with caffeine at 2.1A resolution
5IUB 2016-06-29 2.1 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with compound 12x at 2.1A resolution
6ZDV 2020-09-16 2.13 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with Chromone 5d
5NLX 2017-09-27 2.14 A2A Adenosine receptor room-temperature structure determined by serial millisecond crystallography
5IUA 2016-06-29 2.2 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with compound 12b at 2.2A resolution
6JZH 2019-10-30 2.25 Structure of human A2A adenosine receptor in complex with ZM241385 obtained from SFX experiments under atmospheric pressure
5VRA 2017-12-13 2.35 2.35-Angstrom In situ Mylar structure of human A2A adenosine receptor at 100 K
5K2B 2016-09-21 2.5 2.5 angstrom A2a adenosine receptor structure with MR phasing using XFEL data
5K2A 2016-09-21 2.5 2.5 angstrom A2a adenosine receptor structure with sulfur SAD phasing using XFEL data
6AQF 2018-01-10 2.51 Crystal structure of A2AAR-BRIL in complex with the antagonist ZM241385 produced from Pichia pastoris
5WF5 2018-02-21 2.6 Agonist bound human A2a adenosine receptor with D52N mutation at 2.60 A resolution
4UHR 2015-04-08 2.6 Thermostabilised HUMAN A2a Receptor with CGS21680 bound
2YDV 2011-05-18 2.6 Thermostabilised HUMAN A2a Receptor with NECA bound
5OLH 2018-01-17 2.6 Structure of the A2A-StaR2-bRIL562-Vipadenant complex at 2.6A obtained from in meso soaking experiments.
4UG2 2015-04-08 2.6 Thermostabilised HUMAN A2a Receptor with CGS21680 bound
3EML 2008-10-14 2.6 The 2.6 A Crystal Structure of a Human A2A Adenosine Receptor bound to ZM241385.
6S0Q 2020-07-15 2.65 Structure of the A2A adenosine receptor determined at SwissFEL using native-SAD at 4.57 keV from 50,000 diffraction patterns
3VG9 2012-02-01 2.7 Crystal structure of human adenosine A2A receptor with an allosteric inverse-agonist antibody at 2.7 A resolution
3QAK 2011-03-09 2.71 Agonist bound structure of the human adenosine A2a receptor
5JTB 2017-05-31 2.8 Crystal structure of the chimeric protein of A2aAR-BRIL with bound iodide ions
5N2R 2017-07-26 2.8 Crystal structure of stabilized A2A adenosine receptor A2AR-StaR2-bRIL in complex with PSB36 at 2.8A resolution
5WF6 2018-02-21 2.9 Agonist bound human A2a adenosine receptor with S91A mutation at 2.90 A resolution
2YDO 2011-05-18 3.0 Thermostabilised HUMAN A2a Receptor with adenosine bound
5OLO 2018-01-17 3.1 Structure of the A2A-StaR2-bRIL562-Tozadenant complex at 3.1A obtained from in meso soaking experiments.
3VGA 2012-02-01 3.1 Crystal structure of human adenosine A2A receptor with an allosteric inverse-agonist antibody at 3.1 A resolution
5UVI 2017-05-24 3.2 Serial Millisecond Crystallography of Membrane and Soluble Protein Micro-crystals using Synchrotron Radiation
3UZA 2012-03-21 3.27 Thermostabilised Adenosine A2A receptor in complex with 6-(2,6-Dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-3-amine
3PWH 2011-09-07 3.3 Thermostabilised Adenosine A2A Receptor
3REY 2011-09-07 3.31 Thermostabilised adenosine A2A receptor in complex with XAC
3UZC 2012-03-21 3.34 Thermostabilised Adenosine A2A receptor in complex with 4-(3-amino-5-phenyl-1,2,4-triazin-6-yl)-2-chlorophenol
5G53 2016-08-03 3.4 Structure of the adenosine A2A receptor bound to an engineered G protein
5UIG 2017-02-08 3.5 Crystal structure of adenosine A2A receptor bound to a novel triazole-carboximidamide antagonist
3RFM 2011-09-07 3.6 Thermostabilised adenosine A2A receptor in complex with caffeine
6GDG 2018-05-16 4.11 Cryo-EM structure of the adenosine A2A receptor bound to a miniGs heterotrimer
6MH8 2019-04-24 4.2 High-viscosity injector-based Pink Beam Serial Crystallography of Micro-crystals at a Synchrotron Radiation Source

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