Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 322 (5905), 1211-7

The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist

Affiliations

The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist

Veli-Pekka Jaakola et al. Science.

Abstract

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

Figures

Figure 1
Figure 1
Crystal structure of A2A-T4L-ΔC. A. Overall topology of A2A-T4L-ΔC. The transmembrane part of A2A-ΔC structure is colored brown (helices I - VIII) and the T4L is in cyan. The structure is viewed perpendicular to the plasma membrane. ZM241385 is colored light blue and the four lipid molecules bound to the receptor are colored as red. The four disulfide bonds are yellow. The sulfate ions are omitted. The extracellular loops (ECL1-3) are colored as green and the intracellular loops are colored as blue. The membrane boundaries are adapted from the OPM database (http://opm.phar.umich.edu/) using β2AR-T4L (2RH1) as a model. B. Rotated 180° around the x-axis. The images were created with PyMOL.
Figure 2
Figure 2
Ligand binding characteristics of A2A-WT, A2A-T4L and A2A-T4L-ΔC. A. Saturation binding isotherm for the binding of [3H]ZM241385 to different A2A-WT, A2A-T4L or A2A-T4L-ΔC receptors confined in membranes of Sf9 cells. The indicated preparations of A2A receptors were incubated with different concentrations of [3H]ZM241385 in the absence (filled shapes and solid lines) and presence (open shapes and dashed lines) of 1 M NaCl as described in SOM. The figure shown represents data combined from two separate experiments performed in triplicate. The equilibrium constant (Kd) values of [3H]ZM241385 in the absence and the presence of 1 M NaCl were 2.1 ± 0.7 nM, 1.3 ± 0.2 nM for A2A-WT; 2.0 ± 0.3 nM, 0.9 ± 0.1 nM for A2A-T4L and 1.8 ± 0.2 nM, 1.0 ± 0.1 nM for A2A-T4L-DC, respectively. B. One point binding assay demonstrating the binding of [3H]ZM241385 to membranes (5 μg / assay point) of HEK 293T cells transfected with A2A-WT, A2A-T4L or A2A-T4L-ΔC. [3H]ZM241385 was used at a concentration equivalent to the previously observed equilibrium constant (Kd). Lower panels - the ability of increasing concentrations of C. the agonist CGS21680 or D. the antagonist ZM241385 to compete with [3H]ZM241385 binding at A2A-WT (circles), A2A-T4L (triangles), A2A-T4L-ΔC (squares) constructs in HEK293T cells was tested in the absence (filled shapes and solid lines) or presence (open shapes and dashed lines) of 1 M NaCl. The figure shown represents data combined from three separate experiments performed in duplicate.
Figure 3
Figure 3
Slight changes in helical positions alter the orientation of the ligand binding pocket. A. A surface rendering of the binding pocket for ZM241385 in the A2A adenosine receptor. Helical positions for A2A adenosine (tan), β2AR (pdbid: 2RH1) (blue) and rhodopsin (pdbid: 1U19) (green) are shown after alignment with the FatCat server (38). Ligands for each receptor are shown to illustrate the differences in binding orientation and the differences in the adenosine A2A binding pocket. B. A top view of the helical bundle illustrating the maximal helical positional shifts of A2A relative to β2AR.
Figure 4
Figure 4
Normalized occluded surface (NOS) area changes due to ligand binding. Increases in occluded surface area are represented as thickened red areas of the protein backbone chain (38). A. Rhodopsin (pdbid: 1U19) with retinal (orange) is shown along with the position of ZM241385 (green) for comparison. Retinal makes extensive contact with helices III, V, VI and VII deep in the binding pocket. B. β2AR bound to carazolol (orange) (pdbid: 2RH1) is shown along with the position of ZM241385 (green) for comparison. Carazolol also makes extensive contacts with helices III, V, VI and VII deep in the binding pocket but is responsible for minimal changes in NOS of Trp 2866.48 the canonical “toggle switch”. C. A2A adenosine receptor bound to ZM241385 (orange carbon) has a very different binding orientation relative to rhodopsin and β2AR having minimal interaction with helices III and V, but extensive interactions with helices VI and VII as well as residues in a ECL2 and ECL3. ZM241385 also forms significant contacts with Trp2466.48. All interacting positions on the receptor are displayed as thick red areas and labeled by their corresponding Ballesteros-Weinstein designation (33).
Figure 5
Figure 5
A Comparison of interactions between helix III (E/DRY motif) and ICL2 for human A2A-T4L-ΔC, human β2AR-T4L (pdbid: 2RH1) and turkey β1AR (pdbid 2VT4). A. A2A-T4L-ΔC interactions. The DRY motif does not participate in any stabilizing ionic interactions similar to β2AR and β1AR. Instead Arg1023.50 may play a role in shifting the pKa of the adjacent Asp1013.49 allowing this residue to make stronger hydrogen bonding interactions with helix II and ICL2. B. Turkey β1AR participates in similar interactions as A2A-T4L-ΔC without the hydrogen bond to helix II. C. β2AR does not contain a helical segment in ICL2 and has a modified set of interactions. D. The canonical “ionic lock” in rhodopsin.
Figure 6
Figure 6
Ligand binding cavity of A2A-T4L-ΔC with ZM241385 bound. A. Residues within 5 Å of the ZM241385 are shown in stick representation. Nitrogen atoms are colored blue, oxygen atoms are colored red, and sulfur atoms are colored yellow. Only the interacting helices, ECL3 and the interacting part of ECL2 are shown. The two disulfide bridges in close proximity to the binding cavity are shown as orange sticks. ZM241385 is positioned co-linear with respect to the transmembrane helices V, VI and VII, and the binding cavity is elongated to the ECL3 and helical ends of TM VI and VII. For comparison to retinal chromophore or beta-blockers binding site, see figure 3 for details. The Phe1685.29 from ECL2 forms various aromatic stacking interactions with the bicyclic core of ZM241385. Trp2466.48 associated with stabilizing the antagonist structure is at 3 Å distance from the furan ring of ZM241385. The binding cavity includes four ordered water molecules shown as light blue dots. B. Schematic representation of the interactions between A2A-T4L-ΔC and ZM241385 at the ligand binding cavity combined with mutation analysis for adenosine agonist/antagonists interactions. Mutations that are reported to disrupt antagonist and/or agonist binding are within blue squares: Glu1695.30, His2506.25, Asn2536.55 and Ile2747.39.

Similar articles

See all similar articles

Cited by 646 articles

See all "Cited by" articles

Publication types

Associated data

Feedback