Research Article

Structure of an Agonist-Bound Human A2A Adenosine Receptor

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Science  15 Apr 2011:
Vol. 332, Issue 6027, pp. 322-327
DOI: 10.1126/science.1202793

Abstract

Activation of G protein–coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A2A adenosine receptor (A2AAR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A2AAR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A2AAR and its ligand. The results define the molecule UK-432097 as a “conformationally selective agonist” capable of receptor stabilization in a specific active-state configuration.

G protein–coupled receptors (GPCRs) are critical cellular signal transduction gatekeepers for eukaryotic organisms. Some GPCRs are activated by ligands that act as agonists; others are inactivated by inverse agonists and antagonists. Efforts to elucidate the crystal structures of GPCRs bound to diffusible ligands have recently yielded structures for five class A (rhodopsin-like) GPCRs: the β2 (17) and β1 (8, 9) adrenergic receptors (β2AR and β1AR), the A2A adenosine receptor (A2AAR) (10), the CXCR4 chemokine receptor (11), and the D3 dopamine receptor (12). All of these structures display a common seven-transmembrane (7TM) topology for GPCRs, as well as substantial variations in functionally divergent regions—especially on the extracellular side of the receptor, which is responsible for the recognition of a vast variety of ligands. To overcome the challenge of crystallizing highly dynamic receptors, each of these recent studies used GPCRs that were engineered by fusions or mutations to increase their stability and were cocrystallized with stabilizing ligands.

The mechanism of GPCR activation by native ligands (or synthetic agonists), however, is a fundamental question that remains largely unsolved. An initial model for GPCR activation was provided by the crystal structures of retinal-free opsin (13, 14), but the absence of any agonist precludes direct generalization of this result to other GPCRs activated by diffusible ligands. To shed light on the mechanism of ligand-induced GPCR activation, we have determined a 2.7 Å crystal structure of the human A2AAR in complex with the A2AAR agonist 2-(3-[1-(pyridin-2-yl)piperidin-4-yl]ureido)ethyl-6-N-(2,2-diphenylethyl)-5′-N-ethylcarboxamidoadenosine-2-carboxamide (UK-432097). This highly potent and selective agonist, which was developed as a drug candidate for chronic obstructive pulmonary disease (COPD) treatment (15), represents a substituted derivative of the native ligand adenosine and a series of other prototypical adenosine receptor agonists such as NECA, CGS21680, ATL-146e, and CI-936 (1618). The availability of both agonist- and antagonist-bound A2AAR structures now provides the opportunity to resolve the basic question of how ligand binding at the extracellular side of the receptor triggers conformational changes at the intracellular side, where G protein and other effectors bind and initiate the cascade of downstream signaling pathways.

Agonist UK-432097 and its binding cavity. The compound UK-432097 was characterized as a full A2AAR agonist (1921) (figs. S1 and S2), and its structure in complex with engineered human A2AAR (A2AAR–T4L-∆C) was determined similarly to that of A2AAR–T4L-∆C bound to the antagonist ZM241385 at 2.7 Å resolution (10, 22) (table S1). UK-432097 (778 daltons) is more than twice as large as ZM241385 and occupies most of the A2AAR ligand-binding cavity (Fig. 1A and fig. S3). The ligand-binding cavity reveals an extensive ligand-receptor interaction network, including 11 hydrogen bonds, one aromatic stacking interaction, and a number of nonpolar (van der Waals) interactions (Fig. 1B and table S2). These interactions explain the high binding affinity and subtype selectivity of this compound and its strong stabilizing effect on A2AAR (table S3).

Fig. 1

Ligand UK-432097 and its binding cavity. (A) Overall view of the ligand-binding cavity. The transmembrane part of A2AAR is shown as ribbon and colored gray (helices I to VII). Ligand UK-432097 is shown as green stick. For comparison, ligand ZM241385 (gray stick) is also shown at the position when the two complex structures were superimposed. Binding cavity is represented by orange surface (calculated with the program Hollow). Part of helix VII is removed for clarity. (B) Schematic representation of the hydrogen-bonding interactions (green dashed lines) between A2AAR and UK-432097. (C) Molecular interactions within the ligand-binding cavity for UK-432097 around the adenine moiety. Interacting residues are shown as sticks with side chains colored orange. Hydrogen bonds between A2AAR and ligand are shown as black dotted lines. (D) Molecular interactions around the ribose moiety. (E) Molecular interactions around the ligand substitution sites. A water molecule is shown as a purple sphere. In (B) to (E), residue mutations reported to disrupt agonist and antagonist binding are indicated with orange squares; residue mutations disrupting only agonist binding are indicated with cyan squares. The images were created with PyMOL.

The bicyclic adenine core of UK-432097 is a common scaffold and is present in nearly all major types of adenosine receptor agonists (e.g., NECA, CGS21680, and other nucleosides) and many antagonists (23). This moiety aligns to the triazolotriazine core of ZM241385 when the two complex structures are superimposed together (Fig. 1A), as predicted in modeling studies (24, 25). The molecular interactions that anchor ZM241385 in this region are also conserved in the UK-432097 binding cavity, including aromatic stacking with Phe168 in extracellular loop 2 (ECL2), nonpolar interaction with Ile2747.39, and two hydrogen bonds with Asn2536.55 (26, 27) (Fig. 1C). Disrupting effects on both agonist and antagonist binding are observed upon mutation of these residues: Phe168 (moderate decrease), Ile2747.39 (major decrease), or Asn2536.55 (complete loss) (25, 28).

The ribose ring is a key feature of almost all known adenosine receptor agonists that differentiates them from corresponding antagonists. In the A2AAR–UK-432097 complex, the ribose moiety of the ligand inserts deeply into a predominantly hydrophilic region of the binding cavity (Fig. 1), with the 2′ and 3′ –OH groups both making hydrogen bonds with His2787.43 (2.8 Å and 3.1 Å, table S2). The 3′ –OH is further anchored by a hydrogen-bonding interaction with Ser2777.42 (3.0 Å) (Fig. 1D). Mutation of these two residues to alanine abolishes high-affinity binding of A2AAR agonists (28). His2506.52 forms a hydrogen-bonding interaction with the carbonyl O4 (3.1 Å) that was not predicted in agonist docking to inactive states of the adenosine receptors. Mutation to alanine or phenylalanine disrupts agonist binding, indicating this residue is important for agonist recognition (29). The ribose 5′-N-ethyluronamide substitution in UK-432097, as well as in NECA and many other adenosine receptor agonist chemotypes, is known to provide additional potency. The N2 of this moiety makes a hydrogen bond with Thr883.36 (3.0 Å), in accord with reduction in binding observed for the T88A mutant of A2AAR (30). Besides these polar interactions, the ribose part of the ligand has close contacts with Val843.32, Leu853.33, Trp2466.48, Met1775.38, and Leu2496.51. Previous mutagenesis data on V84A or L249A suggested that these nonpolar interactions are essential for agonist binding (25, 29).

The bulky 2-(3-[1-(pyridin-2-yl)piperidin-4-yl]ureido)ethylcarboxamido substitution in UK-432097 is located at the adenine C2 position; similar extended chains are found in many of the selective A2AAR agonists under development (15). The carboxamide moiety forms an indirect water-mediated hydrogen bond with Tyr91.35 and the carbonyl group of Ala632.61, which contributes to a closer contact of the ligand with helices I and II (Fig. 1E). The urea group is coordinated on both sides by Glu169 (ECL2) forming two hydrogen bonds at N8 and N9 positions (2.8 Å and 2.7 Å) and Tyr2717.36 forming one hydrogen bond at O6 (2.7 Å). Finally, the pyridinyl-piperidine moiety leans to the two phenyl rings and bulges toward helix VII and ECL3. This combined moiety might impose an allosteric effect on structural and conformational changes of the receptor and contribute to subtype binding selectivity (18, 3133).

Antagonist-bound to agonist-bound transformations in A2AAR. A comparison of the A2AAR complexes with antagonist ZM241385 and agonist UK-432097 shows that although the core adenine interactions are conserved between the two structures, binding of the agonist UK-432097 triggers a series of conformational changes within the binding cavity of A2AAR (Fig. 2). Importantly, some of these modest local changes promote large-scale rearrangements in the 7TM helical bundle (Fig. 3). The specific molecular interactions with the ribose moiety of UK-432097 are likely a common feature for all A2AAR agonists, with motions of the ribose-coordinating residues related to the receptor activation. In helix VI, for example, His2506.52 moves ~1.8 Å inward to a position that would otherwise clash with the furan ring of ZM241385 and forms a hydrogen bond with the carbonyl group (Fig. 1D and Fig. 2A). The conserved Trp2466.48 indole moves ~1.9 Å to avoid a steric clash with the ribose ring of the agonist, a movement that facilitates rotation and tilt of the intracellular side of helix VI below Pro2486.50. Although helix V residues Met1775.38, Asn1815.42, and Val1865.47 make only limited nonpolar contacts with the agonist, the movement of His2506.52 allows an inward shift of Val1865.47, promoting an inward tilt of the whole intracellular side of helix V (Fig. 2A).

Fig. 2

Ligand-binding cavity comparison between A2AAR–UK-432097 and A2AAR-ZM241385 complexes. Two receptors were superimposed using Cα atoms of the TM helices. UK-432097–bound A2AAR is colored orange (ribbon) with ligand in green (stick); ZM241385-bound A2AAR (PDB ID 3EML) is colored yellow (ribbon) with ligand in gray (stick). (A) Comparison of the binding pocket around the ligand ribose moiety. Deviating residues (with either backbone or side-chain movement) are shown as sticks. The direction of movement is indicated by black arrows. For clarity, only helices III, VI, VII, and part of helix V are shown. (B) Binding pocket comparison around the substitution part of the ligand. Prominent rotameric switches of Glu169 and His264 on ECL2 and ECL3, respectively, are indicated by dotted black arrows. The images were created with PyMOL.

Fig. 3

Analysis of conformational variations between the A2AAR–UK-432097 complex (orange) and the A2AAR-ZM241385 complex (yellow; PDB ID 3EML). Two protein conformations were superimposed using Cα atoms of the TM helices. Heavy-atom RMSDs were calculated for each residue of the receptor. (A) Side view of helices I to IV. (B) Side view of helices V to VIII. (C) Extracellular (top) view. (D) Intracellular (bottom) view. Most pronounced global changes in the TM helices are shown by black arrows. Side chains that change rotamer conformation are shown as sticks. (E) Plot showing individual residue deviations between superimposed A2AAR conformations, where each residue dot is also colored by deviation of its Cα atom (blue-green dots with high RMSD thus represent residues with rotameric switches). Regions of intracellular loops and helix VIII in the plot are shaded blue; the extracellular loops are shaded pink. The images were created with ICM software (Molsoft LLC). Movie S1 is an animated version of this figure.

In helix VII, Ser2777.42 and His2787.43 move about 2 Å closer to helix III and together with the side chain of Thr883.36 form a hydrogen-bonding network coordinating the ribose moiety (Fig. 1D and Fig. 2A). Two residues on the extracellular end of helix VII, Leu2677.32 and Met2707.35, form additional nonpolar interactions with the two phenyl rings and piperidine rings of UK-432097, resulting in an outward movement of these two residues along with the extracellular part of helix VII by about 2 Å (Fig. 2B). The motion of the helix VII apex correlates with an even more pronounced (3 to 4 Å) outward movement of the adjacent ECL3 backbone, where the His264 imidazole swings by 100° from beneath the pyridine ring to the top of the ligand-binding cavity (Fig. 2B). Also, Glu169 (ECL2) has a different rotamer conformation than in the A2AAR-ZM241385 structure, where its carboxylic group forms a hydrogen bond with the exocyclic amine of ZM241385. Because of a substitution in the exocyclic amino group of UK-432097, the Glu169 carboxyl group moves about 4 Å to form hydrogen bonds with the urea substituent instead, coordinating both the urea group and the two phenyl rings in position. The latter two changes in ECL3 and ECL2 largely depend on substituted moieties in the agonist and are likely to be specific for UK-432097 or agonists with similar bulky substitutions.

In helix III, coordination of the ribose ring through hydrogen bonding interaction with Thr883.36 side chain, as well as nonpolar contacts with Val843.32 and Leu853.33, requires a ~2 Å shift of these residues, resulting in an upward shift of the entire helix III along the helical axis (Fig. 2A and Fig. 3A).

Comparison of this A2AAR-agonist complex structure to its antagonist-bound form shows that helices I to IV form a stable helical bundle “core” with minimal structural changes, while helices V to VII undergo substantial conformational changes (34). The movements are illustrated in Fig. 3, A to D, by graphical superimposition of the two protein conformations (using Cα atoms of TM helices), as well as by plotting full residue RMSDs (root mean square deviations; shown on y axis) and deviations of Cα atoms (shown by color) for individual residues in Fig. 3E. This “RMSD fingerprint” representation conveniently differentiates side-chain rotameric switches (green-blue outliers, marked by residue labels) and global motions of helices (clusters of yellow-red dots).

One of the most prominent changes associated with the agonist-bound structure involves coordinated movements of the intracellular parts of helices VI and V. While the extracellular part of helix VI is fixed in place by the key interactions of Asn2536.55 with the exocyclic amino group of UK-432097, the agonist-induced shift of the conserved Trp2466.48 promotes an outward tilt of the intracellular part of helix VI by about 3 to 4 Å and clockwise rotation by about 30° (Fig. 3). The shift of helix VI is accompanied by coordinated rotameric switches in Phe2015.62 and Tyr1975.58 side chains. Whereas in A2AAR-ZM241385 the conserved Tyr1975.58 is placed in the middle of the helical bundle between helices III and VI, in the UK-432097–bound form this residue moves outward allowing helix V shift toward helix VI. As a result of this combined tilt, the intracellular ends of helices V and VI move closer together in the A2AAR–UK-432097 structure (~6 Å) as compared to the A2AAR-ZM241385 structure (~8 Å) (Fig. 3D).

Another response to the binding of the agonist UK-432097 is a seesaw-like movement of helix VII around the ribose ring. While interactions of the ribose ring hydroxyls with Ser2777.42 and His2787.43 pull these residues of helix VII inward, strong clashes of the two phenyl rings with Leu2677.32, Met2707.35, Tyr2717.36, and ECL3 residues push the extracellular part of helix VII outward, creating a lever that promotes a tilt of helix VII. The conserved NPxxY motif composed of Asn2847.49, Pro2857.50, Phe2867.51, Ile2877.52, and Tyr2887.53 at the cytoplasmic end of helix VII shifts as much as 4 to 5 Å inward, resulting in reorganizations of these side chains, especially Tyr2887.53 (Fig. 3; see also Fig. 4, A and C).

Fig. 4

Comparison of conformational changes in A2AAR TM bundle between antagonist ZM241385 (yellow; PDB ID 3EML)–bound and agonist UK-432097 (orange)–bound states to conformational changes in rhodopsin between inactive (cyan; PDB ID 1GZM) and active-like (purple; PDB ID 3DQB) states. The proteins were superimposed using Cα atoms of the TM helices. Side chains with switches in rotamer state and those critical for agonist interactions are shown as sticks. (A) Intracellular view on the 7TM helical bundle shown for A2AAR antagonist- and agonist-bound states (left) and for inactive rhodopsin and active-like opsin (right). (B) Comparison of movements in helices V and VI of A2AAR (yellow/orange) and rhodopsin (cyan/purple). (C) Same for movements in helix VII. (D) Plot compares individual residue deviations in the 7TM bundle for A2AAR and rhodopsin GPCRs; each residue dot is also colored by deviation of its Cα atom. Intracellular loops and helix VIII regions are shaded blue; extracellular loops are shaded pink. The images were created with ICM software (Molsoft LLC).

Insight into a GPCR activation mechanism. The conformational changes observed between the A2AAR-agonist and A2AAR-antagonist complexes can be further understood in the context of comparison with the structural transitions in the light-activated rhodopsin-opsin system (13, 14). Unliganded opsin at low pH adopts a conformation similar to light-activated rhodopsin at physiological pH (35). Concomitant structural transitions between the inactive rhodopsin and active-like opsin include side-chain switches in the conserved D[E]RY and NPxxY motifs and rearrangements of helices V to VII. Structural superimposition, along with “RMSD fingerprints” of individual residues, between the two pairs of models (inactive and active A2AAR structures; rhodopsin and opsin structures) reveals considerable similarity attributed to a general GPCR activation mechanism (Fig. 4).

The RMSD fingerprint plots shown in Fig. 4D for both inactive-active pairs (A2AAR antagonist–A2AAR agonist; rhodopsin-opsin) of TM domains illustrate similarities between the conformational transitions in these two different receptor systems. For example, a similar pattern is observed of very low deviations in helices I, II, and IV, and of elevated backbone deviations in helices III, V, and VI [correlation coefficient (R2) between two sets of RMSDs: R2 = 0.64 for helices I to VI, R2 = 0.84 for helix VI alone]. In contrast, the plot suggests a lack of similarity in helix VII (R2 = 0.25 for helix VII alone).

The predominant feature common between opsin and A2AAR is the overall movement of helices V and VI, with their extracellular parts being the least mobile and intracellular parts deviating markedly. The conserved Trp6.48 is an important residue triggering the motion of helix VI during activation in A2AAR; however, contrary to the previous “toggle switch” model, this residue does not undergo a rotamer transition, but rather moves along with the backbone in both opsin and A2AAR. The overall backbone shifts in both opsin and A2AAR are accompanied by rotameric switches of Tyr5.58; however, the observed changes in this residue upon activation are very different: Whereas in opsin this side chain swings from outside toward the axis of the TM bundle, the movement in A2AAR is the opposite (36). Also, rotameric changes in nonconserved rhodopsin side chains Phe5.55 and Phe5.43 were not observed in the same positions of Leu5.55 and Phe5.43 in A2AAR.

Another characteristic molecular switch in the rhodopsin-opsin activation model is a salt bridge (“ionic lock”) between Arg3.50 of the conserved D[E]RY motif in helix III and Glu6.30 in helix VI. The ionic lock breaks in opsin upon activation, resulting in rotameric changes in Glu6.30 and movement of helix VI away from helix III (Fig. 4A). Although the ionic lock is already broken in the A2AAR-antagonist structure (10), the concerted movements of helices III and VI observed in the A2AAR–UK-432097 complex also pull Arg1023.50 and Glu2286.30 farther apart, precluding any potential interactions between these residues. A noteworthy difference in this region is that while in our A2AAR-agonist complex structure the middle segment of the cytoplasmic half of helix VI bulges outward by ~3 to 4 Å, the movement of the intracellular tip of helix VI is not as pronounced as in opsin (~3 Å versus 6 to 7 Å, Fig. 4A). However, such partial “attenuation” of the outward tilt of helix VI in this structure may be attributed to the fused T4 lysozyme (T4L), which is likely to limit mobility of the cytoplasmic ends of helices V and VI.

A third featured transition that may be related to activation is a movement of helix III, sliding along its axis toward the extracellular side by ~2 Å, in the context of the overall well-preserved bundle “core” of helices I to IV (37). This movement can be found in both the rhodopsin-opsin and A2AAR systems; in the case of the A2AAR–UK-432097 complex, it allows formation of new contacts with the ribose ring of the agonist.

Conformational changes in helix VII also reveal both common and distinct features for A2AAR and rhodopsin-opsin (Fig. 4). One common feature is a large-scale (>5 Å) movement and rotameric switch of the Tyr7.53 side chain in the highly conserved NPxxY motif at the intracellular tip of helix VII. In the A2AAR-agonist structure, however, the Tyr2887.53 movement is coordinated by a rigid body tilt of helix VII, so that the Cα atoms in this region move more than 5 Å. Conversely, in opsin the backbone of helix VII does not move overall, whereas movement of Tyr7.53 is achieved via local bulging of the backbone in this region. This conformational change in Tyr7.53 has been implicated in the activation mechanism of rhodopsin and other GPCRs, and is likely to be important for the transition from the inactive to the active state in A2AAR (14, 3840). Note that although the changes described above in A2AAR helices III, V, VI, and VII have a major component of “rigid body” movement, a more detailed analysis shows certain conformational plasticity of the helices. In other words, rather than behaving as “rigid sticks,” the movements of TM helices are more consistent with elastic spring behavior (see also fig. S4).

Agonist-bound structures of β1ARs and β2ARs were recently reported (2, 5, 9). The β1AR structures (9) and the structure of β2AR bound to an irreversible agonist (5) highlighted important conformational changes in the orthosteric binding site, but they did not exhibit agonist-induced conformational changes at the intracellular side of these receptors. The active-like state conformation in β2AR with intracellular conformational changes was achieved by additional stabilization of an agonist-bound receptor with a G protein–mimicking nanobody (2). The nanobody-stabilized, agonist-bound β2AR structure revealed large-scale movements of helices III, V, VI, and VII along with rotameric switches of conserved residues at the cytoplasmic G protein–binding interface, similar to those observed in the activated opsin and in the current agonist-bound A2AAR structure. Some variations in the magnitude of these movements may be partially explained by intrinsic differences between these three receptors, as well as by specific effects of the receptor binding partners. For example, a larger magnitude of helix VII movement in A2AAR may result from conformational changes specific for the bulky UK-432097 ligand, whereas the magnitude of helix VI movement in β2AR may be exaggerated by the nanobody binding. Despite the similar conformational transformations occurring at the intracellular side of the nanobody-stabilized, agonist-bound β2AR and the agonist-bound A2AAR, these changes appeared to be induced by rather disparate triggers associated with agonist binding in different receptors. Whereas ligand-induced activation in β2AR is largely driven by an inward movement of helix V (2, 41), binding of A2AAR agonist does not directly affect helix V; instead, the most pronounced ligand-induced changes involve helices III, VI, and VII.

The observed resemblance in the overall structural rearrangements of A2AAR, opsin, and β2AR suggests common features of activation mechanism in GPCRs (Fig. 5). Although agonist binding at the extracellular domain triggers only subtle conformational changes within the binding pocket, some of these changes propagate toward the cytoplasmic domains, promoting large-scale 7TM rearrangement required for G protein binding and signaling. Movements on helices V to VII are supported by a relatively stable core bundle composed of helices I to IV. Comparison of activation transitions in A2AAR, opsin, and β2AR reveals very different types of molecular triggers in the binding pocket, which promote or stabilize an active state. Further understanding of these specific agonist-induced changes in GPCRs may help to establish a structural basis for the functional selectivity of ligands and benefit the design of better agonist-based pharmaceuticals.

Fig. 5

Modularity and common features of the activation mechanism in class A GPCRs. Relatively small agonist-specific conformational changes in the extracellular ligand-binding module of the receptor (red) promote major rearrangements in the intracellular module (blue). The major changes include pronounced tilt of the intracellular parts of helices V (inward) and VI (outward), the latter combined with rotation, an inward tilt of helix VII, and an axial shift of helix III. Although the magnitude of these changes observed in the three receptors (opsin, β2AR, and A2AAR) varies, the overall direction of movement of the helices and their effect on reshaping of the G protein–binding site are similar. The modularity of GPCR activation mechanism consists of a relatively stable “core” bundle of helices I to IV that changes the least, and a more mobile module comprising helices V to VII. This architecture is reminiscent of antibodies, where a conserved two-domain framework is combined with complementarity-determining regions (CDRs) or CDR loops to recognize a diverse array of molecules.

Previous work had suggested that it would be difficult to obtain an agonist-bound structure in an active state without additional stabilization by a G protein or a mimetic. Studies of a number of different agonists in crystallization trials with different GPCR types had suggested that the agonists increased the dynamics of the receptor, leading to poor-quality crystal x-ray diffraction. Our study now shows that it is possible to obtain agonist-bound structures at high resolution with a careful choice of conformationally selective ligands, such as the agonist UK-432097 that forms an extensive network of ligand-receptor interactions. It appears that agonist binding does not always induce a greater degree of dynamics on the receptor structure. Given that some ligands, such as UK-432097, predominantly stabilize only one receptor conformation (conformationally selective ligands), whereas others shift the dynamic equilibrium of multiple receptor conformations (42), the variety of agonist types may be broader than previously believed.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1202793/DC1

Materials and Methods

Tables S1 to S3

Figs. S1 to S4

References

Movie S1

References and Notes

  1. The ligand-binding properties of UK-432097 and related nucleosides were examined in membranes from A2AAR–T4L-∆C–expressing Sf9 cells (fig. S1). The inhibition constant (Ki) of UK-432097 at the A2AAR–T4L-∆C receptor was 4.75 nM. The agonist property of UK-432097 was characterized with CHO cells expressing human wild-type A2AAR. The efficacy and potency of UK-432097 relative to other three known agonists (NECA, CGS21680, and CI-936) were followed using a cyclic adenosine monophosphate accumulation assay in intact CHO cells. The respective half-maximal effective concentration (EC50) values of UK-432097, NECA, CGS21680, and CI-936 are 0.66 ± 0.19 nM, 5.99 ± 1.86 nM, 3.25 ± 1.22 nM, and 14.5 ± 5.81 nM (fig. S2).
  2. See supporting material on Science Online.
  3. To overcome the inherent structural flexibility of GPCRs, we used the same engineered A2AAR construct, termed A2AAR–T4L-∆C, as was used in the A2AAR-ZM241385 complex structure. Briefly, this construct contains an insertion of T4 lysozyme (T4L) at the intracellular loop 3 (ICL3) and removal of C-terminal 96 residues of A2AAR. Stabilization of the A2AAR–T4L-∆C construct in an active state was achieved by screening a broad range of A2AAR agonists using a thermal stability assay (table S3). Among the tested ligands, UK-432097 exhibited the highest thermal stability (melting temperature ~65°C). The stabilized A2AAR–T4L-∆C in complex with UK-432097 was crystallized in a cholesterol-doped monoolein lipidic cubic phase (LCP). Crystallographic data were collected on the GM/CA-CAT beamline at the Argonne Photon Source using a 10-μm minibeam. A complete data set at 2.7 Å resolution was assembled using data collected from 20 crystals. The final model includes residues Ile3 to Leu308 of the human A2AAR, residues 2 to 161 of T4L replacing the ICL3 of A2AAR (Lys209 to Ala221), two partial lipid [OLC, (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate] chains, six water molecules, and the agonist UK-432097 bound in the ligand-binding cavity. Part of the extracellular loop 2 (ECL2; Pro149 to Gln157) was not modeled because of a weak electron density.
  4. In Ballesteros-Weinstein numbering, a single most conserved residue among the class A GPCRs is designated x.50, where x is the transmembrane helix number. All other residues on that helix are numbered relative to this conserved position. Numbers decrease toward the N terminus and increase toward the C terminus. This numbering system is used as superscript only for labeling residues on transmembrane helices (but not for those on loop regions). For clarity, residues for A2AAR use both standard and Ballesteros-Weinstein numberings, whereas residues for other GPCRs use only Ballesteros-Weinstein numbering throughout this text.
  5. Note that global superimposition of TM helical bundle in this case almost exactly overlaps common features of A2AAR-ZM241385 and A2AAR–UK-432097 complexes, so that the Asn2536.55 α-carbonyls of A2AAR are within 0.2 Å and exocyclic amines of ZM241385 and UK-432097 are within 0.6 Å distance.
  6. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (K.A.J. and Z.-G.G.), PSI:Biology grant U54 GM094618 for structure production, and NIH Roadmap Initiative grant P50 GM073197 for technology development. R.C.S. thanks N. Dekker at AstraZeneca for suggesting the UK-432097 compound for biochemical and structural studies. We thank J. Velasquez for help on molecular biology; C. Cornillez-Ty, T. Trinh, and K. Allin for help on baculovirus expression; E. Chien and W. Liu for advice on protein purification and LCP crystallization; I. Wilson, M. Hanson, and A. IJzerman for careful review and scientific feedback on the manuscript; K. Kadyshevskaya for assistance with figure preparation; A. Walker for assistance with manuscript preparation; L. Heitman for A2AAR compounds used in thermal stability studies; Y. Zheng and M. Caffrey for use of an in meso robot (built with support from NIH grant GM075915, NSF grant IIS0308078, and Science Foundation Ireland grant 02-IN1-B266); and J. Smith, R. Fischetti, and N. Sanishvili for assistance in development and use of the minibeam and beamtime at GM/CA-CAT beamline 23-ID at the Advanced Photon Source, which is supported by National Cancer Institute grant Y1-CO-1020 and National Institute of General Medical Sciences grant Y1-GM-1104. R.C.S. is a founder and member of the board of directors of Receptos, a GPCR structure–based drug discovery company. Atomic coordinates and structure factors have been deposited in the Protein Data Bank with identification code 3QAK.
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