Crystal structure of the adenosine A_2Areceptor 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/<i>N</i>-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.