Extracellular adenosine plays an important role in physiology and initiates most of its effects through the activation of four guanine nucleotide binding protein (G protein) coupled receptor (GPCR) subtypes, A₁, A_2A, A_2B and A₃ (1, 2). Each of these four receptors plays an essential role in responding to adenosine in the central nervous system (3, 4) regulating pain (5), cerebral blood flow (6), basal ganglia functions (7), respiration (8) and sleep (9). These receptor subtypes are primarily coupled to the cAMP second messenger system and each has its own unique pharmacological profile. The A_2A adenosine subtype is linked to G_s and G_olf proteins and upon receptor activation, the intracellular levels of cAMP increase. At least three of the four adenosine receptor subtypes (A₁, A_2A and A_2B) are blocked by naturally occurring methylxanthines, such as caffeine, with modest affinity. Interestingly, strong epidemiological evidence suggests that coffee drinkers have a lower risk of Parkinson’s disease (10). This effect has been linked to caffeine’s interaction with the A_2A adenosine receptor, which controls locomotor behavior in basal ganglia together with dopamine D₂ and metabotropic glutamate mGluR receptors (11, 12). Development of more selective compounds for adenosine receptor subtypes could provide a class of therapeutics for treating numerous human maladies, such as pain (5), Parkinson’s disease (7, 13), Huntington disease (14), asthma (15), seizures (16) and many other neurological disorders (14, 17).

GPCRs possess numerous thermodynamic conformations (20, 21), implying an inherent structural flexibility (22-24). This flexibility manifests itself as thermal instability upon detergent extraction from lipid membranes and is one of the primary challenges in generating crystals of GPCRs (25, 26). In order to overcome this obstacle with the human A_2A adenosine receptor, we applied the T4L fusion strategy (24, 27, 28), where most of the third cytoplasmic loop (Leu209^5.70 - Ala221^6.23) was replaced with lysozyme from T4 bacteriophage and the carboxyl-terminal tail (Ala317 - Ser412) was deleted to improve the likelihood of crystallization. The resulting recombinant construct (A_2A-T4L-ΔC) was further stabilized during purification with (i) sodium chloride, which has a beneficial effect on adenosine receptor stability, (ii) a saturating concentration of the nonspecific adenosine receptor antagonist theophylline (ZM241385 was exchanged from theophylline in the last purification step) and (iii) including cholesteryl hemisuccinate throughout the purification. Purified A_2A-T4L-ΔC bound to ZM241385 was crystallized using the in meso crystallization methodology where the lipid phase consisted of a mixture of monoolein and cholesterol (29).

The residues constituting the transmembrane α-helices are: Gly5^1.31-Trp32^1.58 (helix I); Thr41^2.39-Ser67^2.65 (helix II); His75^3.23-Arg107^3.55 (helix III); Thr119^4.40-Leu140^4.61 (helix IV); Asn175^5.36-Ala204^5.65 (helix V); Arg222^6.24-Phe258^6.60 (helix VI); Leu269^7.34-Arg291^7.56 (helix VII) (33). A small non-transmembrane helix is located at the membrane-cytoplasm interface and comprises Arg296^8.47-Leu308^8.59 (helix VIII). The A_2A adenosine receptor does not contain the canonical palmitoylation site(s) found in the majority of GPCRs; instead, helix VIII is stabilized by interactions with helix I (34). The residues defining intracellular and extracellular loops (ICLs and ECLs) are: Leu33^1.59-Val40^2.38 (ICL1); Ile108^3.56-Gly118^4.39 (ICL2); Leu208^5.69-Ala221^6.23 (ICL3); Thr68^2.66-Cys74^3.22 (ECL1); Leu141^4.62-Met174^5.35 (ECL2); Cys259^6.61-Trp268^7.33 (ECL3). In our structure ICL3 has been replaced by 160 residues from T4L lysozyme (see Supplementary Figure S1). Additionally, The N-linked glycan associated with Asn154^4.75 has been removed enzymatically to improve crystallization.

A common feature of the class A GPCRs is the presence of a tryptophan residue (at position 6.48) on helix VI whose rotameric position is thought to control the equilibrium between the active and inactive states of each receptor (45). Based on the position of retinal in the rhodopsin structure it was proposed that ligand interactions with this key residue could modulate receptor equilibrium (46). Interestingly, the contact area between ligand and the “toggle switch” tryptophan residue at position 6.48 varies considerably among the solved receptor structures. For instance, rhodopsin and β₂AR have a similar binding mode as noted; however, retinal in rhodopsin has a contact area of 36 Ų, whereas carazolol bound to β₂AR lacks any direct contact with Trp286^6.48 (27). Ground-state rhodopsin has virtually no basal activity, whereas β₂AR has a relatively high basal activity which is suppressed somewhat by carazolol as an inverse agonist (23, 47). The observed increase in contact area may have direct implications for inverse agonist efficacy or suppressed basal activity by limiting the range of motion of the “toggle switch” tryptophan. The competitive antagonist ZM241385 has a 14 Ų contact area with Trp246^6.48 despite an altered binding mode relative to rhodopsin (Figure 4c). Although it remains to be established biochemically, this finding is suggestive of the ability of this ligand to stabilize the A_2A adenosine receptor in an inactive state.

To date, structural and biophysical data on the class A GPCRs with diffusible ligands has been dominated by the biogenic amine receptors, such as the adrenergic, dopamine, and serotonin families. These amine ligands are all positively charged at physiologic pH and are known to interact with a key negatively charged aspartate residue (Asp^3.32) on helix III. Indeed, in all three of the available β-adrenergic structures, each co-crystallized ligand interacts with this residue and binds in a pocket quite similar to that of retinal in rhodopsin. Analysis of the binding sites of the three available GPCR structures indicates two possibilities for the other members of the class A family: i) other ligands will bind in a spatially similar binding site, with the ligand specificity dictated by sequence differences within the binding pocket, or ii) ligands for other receptors may bind in a completely different fashion interacting with other positions on the receptor. In contrast to the β-adrenergic ligands and retinal, ZM241385 occupies a significantly different position in the transmembrane network (Figure 4) where its orientation is almost perpendicular to the membrane plane (Figures ​(Figures4c4c and ​and6).6). The bicyclic triazolotriazine core of ZM241385 is anchored by an aromatic stacking interaction with Phe168^5.29, an aliphatic hydrophobic interaction with Ile274^7.39 and a hydrogen bonding interaction with Asn253^6.55 (Figure 6). Adjacent to Phe168^5.29 a polar residue (Glu169^5.30) interacts with the exocyclic amino group (N15 atom) linked to the bicyclic core of ZM241385 (Figure 6b). Mutation of Glu169^5.30 to alanine reduces the affinity for both antagonists and agonists and causes a 1000-fold reduction in agonist efficacy (54). However, mutating this position to glutamine did not have a substantial impact on antagonist binding affinity, suggesting hydrogen bonding as the predominant means of interacting with N15 of ZM241385 as opposed to Coulombic interactions (Figure 6b). Early studies indicate that mutation of Asn253^6.55 to alanine, which would disrupt an important polar contact with the exocyclic N15 atom of ZM241385, results in a complete loss of both agonist and antagonist binding (55). The structure also shows that Ile274^7.39 forms a hydrophobic contact with the C12 atom of ZM241385; accordingly mutation of Ile274^7.39 to alanine results in negligible antagonist binding and a 30-fold reduction in agonist potency (55). No mutagenesis data is available for Phe168^5.29 or Leu249 both of which anchor the bicyclic ring of ZM241385 through π stacking and hydrophobic interactions respectively although their involvement in ligand binding has been proposed (56). The phenolic hydroxyl group extending from the ethylamine chain of ZM241385 forms a hydrogen bond with an ordered water molecule. The phenyl ring forms hydrophobic interactions with Leu267^7.32 and Met270^7.35 that would suggest hydrophobicity rather than aromaticity as means of interaction with the phenolic substituent. Indeed, a ZM241385 derivative, with a cycloalkyl substituent (LUF5477) instead of phenylmethylene, also has high affinity for the A_2A adenosine receptor. In a recent study on new antagonists for the A_2A adenosine receptor it was demonstrated that tremendous substituent flexibility exists in this area of the pharmacophore (57). This observation correlates well with the directionality of the phenylethylamine substituent in ZM241385 as it is directed towards the more solvent exposed extracellular region (ECL2 and ECL3) rather than towards the transmembrane domain of the receptor as was previously proposed (38, 39). The other substituent in ZM241385 is the furan ring, a feature that occurs in many A_2A adenosine receptor antagonists. This moiety is located deep in the ligand binding cavity and directed towards helices V and VII, where it hydrogen bonds to Asn253^6.55 and forms a water-mediated interaction with His250^6.52 (Figure 6A). Hydrophobic interactions of the furan ring system include His250^6.52 with C23 and Leu249^6.51 with the C22 and C21 atoms of ZM241385. Mutation of His250^6.52 to alanine completely abolishes ligand binding, whereas mutation to phenylalanine or tyrosine residues modestly affects agonist binding but not antagonist binding (55, 58); replacement with an asparagine slightly increases ligand affinity (58). The furan ring is approximately 3 Å away from the highly conserved Trp246^6.48 an important residue in receptor activation as discussed above (59). We speculate that the hydrophobic interactions between ZM241385’s furan ring and this residue will hinder the structural rearrangements necessary for activation, constraining the receptor in an inactive state.