Cryo-EM structures of adenosine receptor A3AR bound to selective agonists

The adenosine A3 receptor (A3AR), a key member of the G protein-coupled receptor family, is a promising therapeutic target for inflammatory and cancerous conditions. The selective A3AR agonists, CF101 and CF102, are clinically significant, yet their recognition mechanisms remained elusive. Here we report the cryogenic electron microscopy structures of the full-length human A3AR bound to CF101 and CF102 with heterotrimeric Gi protein in complex at 3.3-3.2 Å resolution. These agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays reveal the critical role of extracellular loop 3 in A3AR’s ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR, significantly impact receptor activation. Comparative analysis with the inactive A2AAR structure highlights a conserved receptor activation mechanism. Our findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.

Residues lining the orthosteric binding site are highlighted with red circles and annotated with GPCR Ballesteros-Weinstein numbering scheme.The C-terminus of the adenosine receptors were omitted.a-e The binding cavities of the adenosine receptors are depicted as gray surfaces, with the bound ligands shown as sticks.The receptor names and associated PDB codes [2][3][4] are indicated below each model.The unique subpocket in the A3AR is indicated by the red boxes.a sequence alignment of part of TM3 and ICL2 of adenosine receptors.The panel was generated on GPCRdb [5] .The red arrows indicate the unconserved residues.b A1AR-Gαi protein interaction.c A3AR-Gαi protein interaction.d A2AAR-Gαs protein interaction.e A2BAR-Gαs protein interaction.The polar interactions are indicated by black dashed lines.b UD indicates that the activation level is too low to determine pEC50 values.c The expression indicates the cell-surface expression which was relative to the wild type.

Fig. S1
Fig. S1 The expression and purification of A3AR-Gi complex.a. Schematic diagrams of the expression constructs of A3AR and Gβ1 using the NanoBiT tethering approach.A3AR and Gβ1 fused with LgBiT and HiBiT, respectively.b.Sizeexclusion chromatography profile of the CF101-A3AR-Gi complex.c.SDS-PAGE of the arrow indicated peak fraction in (b).d.Size-exclusion chromatography profile of the CF102-A3AR-Gi complex.e. SDS-PAGE of the arrow indicated peak fraction in (d).

Fig.
Fig. S2 Cryo-EM data processing of CF101-A3AR-Gi complex.a. Representative image from cryo-EM dataset.Scale bar, 50 nm.b.Respresentative 2D average classification classes.Scale bar, 10 nm.c.Flow-chart of the cryo-EM data processing.d.FSC curves.e.The local resolution map.

Fig.
Fig. S3 Cryo-EM data processing of CF102-A3AR-Gi complex.a. Representative image from cryo-EM dataset.Scale bar, 50 nm.b.Respresentative 2D average classification classes.Scale bar, 10 nm.c.Flow-chart of the cryo-EM data processing.d.FSC curves.e.The local resolution map.

Fig. S4
Fig. S4 Respresentative regions of cryo-EM density maps are shown for the each transmembrane helical (TM) of A3AR and the α5 and αN helices of Gαi.

Fig. S6
Fig. S6 Effects of CF101 or CF102 on the A3AR mutants using NanoBiT association assay.These residues in A3AR formed hydrophobic interactions with the 3-iodobenzyl group present in CF101 and CF102.Data shown are mean ± S.E.M. of three independent experiments (n = 3).

Fig. S7
Fig. S7 The role of Y15 1.35 and Y265 7.36 .a Y15 1.35 formed hydrophobic interaction with Y265 7.36 in CF102-bound A3AR.b-e Effects of CF101 and CF102 on the A3AR and mutants of Y15 1.35 F and Y265 7.36 A using NanoBiT association assay (b and c) and cAMP accumulation assay (d and e), respectively.Data shown are mean ± S.E.M. of three independent experiments (n = 3).Source data are provided as a Source Data file.

Fig. S9
Fig. S9 The orthosteric binding pockets among adenosine receptors.The positions highlighted indicate where unique residues occurred in A3AR compared to other adenosine receptors.The receptor names and their associated colors were shown above the models.The side chains in A3AR were depicted as bold sticks, while the corresponding side chains in other adenosine receptors were shown as thick sticks.

Fig. S10
Fig. S10 Effects of CF101/CF102 on A3AR mutants containing swapped residues from other adenosine receptors by cAMP accumulation assay.Data shown are mean ± S.E.M. of three independent experiments (n = 3).Source data are provided as a Source Data file.

Fig. S11
Fig. S11 The binding cavities in adenosine receptors.

Table S2 Cell surface expression of A3AR and its mutants on CF101-and CF102- induced NanoBiT assay.
a Data shown are means ± S.E.M. from at least three independent experiments.b UD indicates that the activation level is too low to determine pEC50 values.c NT, not test.*

Table S3 Cell surface expression of A3AR and its mutants on CF101-and CF102- induced cAMP assay.
UD indicates that the activation level is too low to determine pEC50 values.
a Data shown are means ± S.E.M. from at least three independent experiments.b

Table S4 Cell surface expression of A1AR/A2AAR/A2BAR and its relative mutant on CF101-and CF102-induced NanoBiT assay.
a Data shown are means ± S.E.M. from at least three independent experiments.