Ligand Binding and Subtype Selectivity of the Human A2A Adenosine Receptor

Abstract

The crystal structure of the human A 2A adenosine receptor bound to the A 2A receptor-specific antagonist, ZM241385, was recently determined at 2.6-Å resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane, and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand the selectivity of ZM241385 for the human A 2A adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe-168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met-177 3 Ala mutation impeded antagonist but not agonist binding. Finally, the Leu-249 3 Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe-168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent "upper" region of the binding cavity while the "lower" part of the binding cavity is conserved across adenosine receptor subtypes.

Ligand-Dependent Activation and Deactivation of the Human Adenosine A2AReceptor

“…60 Although both Leu249 6.51 and Met177 5.38 consistently interact with the ligands, the Leu249Ala mutation affects both agonist and antagonist binding, while Met177Ala affects binding of antagonist ZMA but not agonist NEC. 56 We explain this difference by comparing the number of nonpolar contacts of these residues with the ligands: Leu249 6.51 on average has 2–6 contacts with all the ligands we studied, in contrast to Met177 5.38 , which interacts tightly with ZMA (3.3 average contacts) but loosely with NEC (1.4 average contacts).…”

“…As presented in Table 1, the residue-ligand interactions are analyzed and categorized into three distinct regions in the binding pocket in accordance with prior experimental studies. 31,55,56 We then relate the propensity of certain residue-ligand interactions to agonism/antagonism, affinity, and selectivity. We believe such information will be beneficial for structure-based design of AA2AR ligands.…”

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition

“…It has been postulated that interactions that determine AR subtype selectivity are localized to the more diverse upper and extracellular regions of the binding pocket [34]. In contrast, the lower portion of the ligand-binding pocket is believed to determine the strength of ligand binding [35]. …”

“…In A 2A R, ECL2 forms a random coil structure with a very short α-helical segment at the end of the loop [3]. This segment could form critical aromatic π-stacking interactions between F168 and the heterocyclic core of various A 2A R agonists and antagonists [3,8,11, 13,35–37]. Additionally, this small α-helical segment contains E169 that could form important polar interactions with various ligands and with H264 in ECL3 [37].…”

From Three-Dimensional GPCR Structure to Rational Ligand Discovery

“…6, 7) (Katritch et al 2010b). Interestingly, specific water molecules in the AA2AR binding site were included in the docking simulations, because retrospective virtual screening evaluations gave better results with water than without consideration of water (Katritch et al 2010b (Kim et al 1995;Jaakola et al 2010;Zhukov et al 2011), in their scoring protocol to rank the docking poses of 1.4 million cpds in the AA2AR crystal structure by increasing the dipole moment of the side chain amide group but without taking water into account (Carlsson et al 2010). Using this experimentally guided SBVS approach, 7 of the 20 selected hits were experimentally confirmed as novel AA2AR ligands, with K i values ranging from 0.2 to 9 M (incl.…”