Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

A cryo-EM structure of the human A3 adenosine receptor bound to the endogenous agonist adenosine (#122)

Liudi Zhang 1 , Jesse Mobbs 1 , Lauren May 1 , David Thal 1
  1. Monash University, Parkville, VICTORIA, Australia

Background: The human A3 adenosine receptor (A3AR) is a G protein-coupled receptor (GPCR) and is one of four similarly related adenosine receptors that are activated by adenosine. The A3AR is highly expressed in inflammatory and cancer cells, and is implicated in numerous diseases including rheumatoid arthritis, psoriasis, dry eye disease, glaucoma, and hepatocellular carcinoma. Given the clinical potential of the A3AR, there is strong interest in drug discovery for novel A3AR therapeutics that includes agonists, antagonists, and allosteric modulators with multiple candidates currently in clinical trials [1, 2]. However, there are no three-dimensional structures of the A3AR, and current approaches for A3AR drug discovery have been heavily reliant on and traditional medicinal chemistry and computational approaches. The determination of high-resolution A3AR structures in complex with antagonists and agonists will greatly aid our understanding of how current A3AR ligands work, and would greatly facilitate future A3AR drug discovery.

Aim: Determine the structure of A3AR in complex with heterotrimeric G protein Gi1 and the endogenous agonist adenosine using cryo-electron microscopy (cryo-EM).

Methods: To determine the structure of the A3AR in complex with Gi1 both proteins were expressed in Sf9 insect cells and solubilised with the detergent LMNG in the presence of adenosine. An A3AR-Gi1 complex was formed by the addition of apyrase and subsequently purified using an anti-FLAG antibody column and size exclusion chromatography. Stabilization of the A3AR-Gi1 complex was further achieved by using a dominant-negative Gi (DNGi2) in the presence or absence of single chain antibody fragment scFv16 [3]. A complex between A3AR, Gi1, and scFv16 was confirmed by SDS using negative stain EM, and cryo-EM data sets were collected on a 200 kV ThermoFisher Artica. 3D refinement is performed in the cryoSPARC (v3.1).

Results: Using cryo-EM, we have determined a 2.9 Å structure of the A3AR-Gi1 in complex with adenosine. Initial analysis reveals significant differences in the conformation of the A3AR in comparison to the A1AR and A2AAR that offers preliminary insight into ligand binding and selectivity.

  1. 1. Borea, P.A., et al., The A3 adenosine receptor: history and perspectives. Pharmacol Rev, 2015. 67(1): p. 74-102. 2. Sachdeva, S. and M. Gupta, Adenosine and its receptors as therapeutic targets: An overview. Saudi Pharm J, 2013. 21(3): p. 245-53. 3. Maeda, S., et al., Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nat Commun, 2018. 9(1): p. 3712. 4. Mukherjee, S., et al., Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins. Nat Commun, 2020. 11(1): p. 1598.