Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

Abstract

The adenosine A_2A receptor (A_2AR) has long been implicated in cardiovascular disorders. As more selective A_2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A_2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A_2A receptor bound to compound 1 (Cmpd-1), a novel A_2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A_2A receptor with a cytochrome b562-RIL (BRIL) fusion (A_2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A_2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A_2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A_2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr9^1.35 and Tyr271^7.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A_2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A_2AR-selective compounds for the treatment of Parkinson's disease.

Keywords: A2A adenosine receptor; GPCR; Parkinson’s disease; allosteric; structure.

Fig. 1.

Cmpd-1 chemical structure and A_2AR–BRIL–Cmpd-1 crystal packing. (A) Chemical structure of several A_2AR ligands, including Cmpd-1, ZM241385, and endogenous ligand adenosine. Cmpd-1 is predicted to be protonated under physiological pH. (B) Crystal packing of the A_2AR–BRIL–Cmpd-1 structure represents an unusual type II crystal packing for a GPCR. The receptor is shown in green, and BRIL is shown in cyan. (C) Schemes of type I and type II packing for membrane protein crystals. Detergent and lipid molecules are shown in pink and gray, respectively. Transmembrane segments and polar region of the membrane protein are shown in green and cyan, respectively.

Fig. S1.

Electron density map and crystal packing of A_2AR–mT4L–Cmpd-1 crystals grown in LCP. (A) Density map of A_2AR–mT4L–Cmpd-1. 2F_o–F_c map is shown in gray mesh at 1.0 σ, and F_o–F_c map is shown in green (for positive peaks) and red (for negative peaks) mesh at 2.0 σ. (B) Close-up view of the potential ligand binding pocket. No density for side chains or the ligand was observed. (C) The crystal packing of A_2AR–mT4L–Cmpd-1 (Left), A_2AR–T4L–ZM241385 (Center), and A_2AR–BRIL–ZM241385 (Right) shown in two orientations 90° apart. The center molecule is colored cyan, and surrounding molecules are colored green.

Fig. S2.

Crystals of A_2AR–BRIL–Cmpd-1 obtained in vapor-phase diffusion. (A) Brightfield and UV images of the crystals grown in vapor-phase diffusion. (Scale bars: Left, 100 μm; Right, 50 μm.) (B) Diffraction pattern of the crystal.

Fig. 2.

Structure features of A_2AR–BRIL in complex with Cmpd-1. (A) Overall structure comparison of A_2AR–BRIL–Cmpd-1 (receptor and ligand in green; BRIL in cyan) and A_2AR–BRIL–ZM241385 (receptor and ligand in orange; BRIL in gold). (B) Structural comparison of the intracellular part of TM5. In the A_2AR–BRIL–Cmpd-1 structure, TM5 is displaced by 8 Å at the Cα of Gln207^5.69 relative to the A_2AR–BRIL–ZM241385 structure. (C) In the A_2AR–BRIL–Cmpd-1 structure, Glu228^6.30 forms a salt bridge with Arg107^3.55, which is only possible because of the displacement of TM5 noted above. The map is 2F_o–F_c map contoured at 1.5 σ. (D) Structural comparison of the N-terminal aspect of the TM1 helices between the two structures where the Cα atoms of Met4^1.30 are ∼4 Å apart.

Fig. S3.

Alternate conformation of Leu167 to Glu169. The involved residues and the ligand Cmpd-1 are shown in sticks. 2F_o–F_c map are shown in blue mesh contoured at 1.0 σ level. The major difference is with Phe168^ECL2. The conformation that allow its aromatic ring to stack with Cmpd-1 is the dominant conformation with the occupancy of 0.7 (shown in green), and the occupancy of the other conformation is 0.3 (shown in gray).

Fig. 3.

Binding interactions between the A_2AR and Cmpd-1. (A) Cmpd-1 binding pocket. The side chains of residues within 4 Å of the ligand are shown in stick. Hydrogen bonding interactions are depicted by dashed lines. The F_o–F_c omit map around the ligand is shown as mesh contoured at 2.5 σ. (B) A schematic representation of Cmpd-1 binding interactions. The residues within 4 Å of Cmpd-1 are shown as spheres, with polar, hydrophobic, and negatively charged residues colored cyan, green, and red, respectively. The π−π stacking interaction of the aminotriazole ring with Phe168 is depicted with a solid green line. The hydrogen bonding interactions with the side chains of Asn253 and Glu169 are depicted with dotted pink lines. The protein pocket is displayed with a line around the ligand, colored according to the nearest residue. A gap in the line shows an opening in the pocket, and the minimum gap observed in this diagram indicates that Cmpd-1 is very well buried. The iminium of Cmpd-1 and His250 are predicted to be protonated due to the low pH (6.5) of crystallization conditions.

Fig. S4.

Comparison of various A_2AR ligands binding positions. The structure of A_2AR–BRIL–Cmpd-1 (green) is superimposed with A_2AR–BRIL–ZM241385 (cyan; PDB ID code 4EIY) and A_2AR-TS–adenosine (salmon; PDB ID code 2YDO). The ligands are shown in stick. The methylphenyl and aminotriazole rings of Cmpd-1 occupy positions that collectively are similar to that of adenosine in the orthosteric site.

Fig. 4.

Potential allosteric site identified in A_2AR–BRIL–Cmpd-1 structure. (A) Comparison of Tyr9^1.35 and Tyr271^7.36 positions and rotamers viewed from extracellular side. The structure of A_2AR–BRIL–Cmpd-1 (green) is superimposed with A_2AR–BRIL–ZM241385 (orange; PDB ID code 4EIY) and A_2AR-TS–ZM241385 (blue; PDB ID code 3PWH). Residues and ligand in A_2AR–BRIL–Cmpd-1 are shown in stick, and they are shown in lines in other structures. (B) The potential allosteric site. The methoxyphenyl ring is located in a compact pocket formed by eight surrounding residues. Cmpd-1 is shown in spheres, and residues are shown in sticks, with the contact residues’ van der Waals radius depicted as dots. (C) Sequence alignment of human adenosine receptors. Residues forming the allosteric pocket are indicated by blue circles. Residues that are strictly conserved among all subtypes of adenosine receptors are highlighted by red shade, and partially conserved residues are highlighted in red characters.

Fig. S5.

Comparison of Tyr9^1.35 and Tyr271^7.36 positions and rotamers with other A_2AR structures. The structure of A_2AR–BRIL–Cmpd-1 (green) is superimposed with A_2AR-T4L–UK-432097 (magenta; PDB ID code 3QAK), A_2AR-TS–adenosine (salmon; PDB ID code 2YDO), A_2AR-TS–NECA (salmon; PDB ID code 2YDV) and A_2ARTS–T4E (yellow; PDB ID code 3UZC). Tyrosine side chains and ligands are shown in stick for A_2AR–BRIL–Cmpd-1 and in lines for the other structures.

Fig. S6.

The outward displacement of TM1 in A_2AR–BRIL–Cmpd-1 structure is incompatible with the crystal packing of A_2AR–BRIL–ZM241385. A_2AR–BRIL–Cmpd-1 (green) is aligned with A_2AR–BRIL–ZM241385 (orange). A symmetry molecule of A_2AR–BRIL–ZM241385 (gray) that packs through their TM1 and TM7 shows steric conflicts with TM1 in A_2AR–BRIL–Cmpd-1 and therefore prevents crystallization in the same lattice as A_2AR–BRIL–ZM241385 in LCP. Residues with steric conflicts are shown in sticks.

Fig. S7.

Comparison of the allosteric pocket in A_2AR–BRIL–Cmpd-1 structure to equivalent parts in other reported A_2AR structures. The structures shown here include A_2AR–BRIL–ZM241385 (cyan; PDB ID code 4EIY), A_2AR-TS–ZM241385 (blue; PDB ID code 3PWH), A_2AR-TS–1,2,4-triazine antagonist (A_2AR-TS-–T4E, yellow; PDB ID code 3UZC), and A_2AR-TS–adenosine (salmon; PDB ID code 2YDO). These structures are all aligned with the A_2AR–BRIL–Cmpd-1 structure (green), and surfaces are all viewed and clipped at same position for comparison. Red arrows point to the equivalent part in each structure and the sites are either very shallow pockets or clefts exposed to solvent.