Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

Abstract

The adenosine A_2A receptor (A_2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein G_S. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A_2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-G_S, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-G_S are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.

Keywords: G protein-coupled receptor; GPCR; adenosine receptors; biochemistry; chemical biology; mini-G protein; molecular biophysics; none; structural biology; structure.

Figure 1.. Pharmacological analyses of A_2AR.

(a) Saturation binding of the inverse agonist ³H-ZM241385 to A_2AR constructs gave the following apparent K_Ds: A_2AR (circles), 0.5 ± 0.1 nM; TrxA-A_2AR (squares), 0.8 ± 0.2 nM. (b) Competition binding curves measuring the displacement of ³H-ZM241385 with increasing concentrations of NECA gave the following K_is for NECA; A_2AR (filled circles), 1.0 ± 0.5 µM; A_2AR + mini-G_S (open circles, dashed line), 2.6 ± 1.8 nM; TrxA-A_2AR (filled squares), 1.1 ± 0.4 µM; TrxA-A_2AR + mini-G_S (open squares, dashed line), 1.8 ± 1.2 nM. Data plotted are the average from two independent experiments performed in duplicate with error bars shown as the SD.

Figure 2.. Cryo-EM of the A_2AR complex in the presence and absence of a VPP.

(a-c) Each panel contains three sections, with the left-hand section showing a representative micrograph obtained on a Titan Krios, the central section depicting 2D class averages and the right-hand section the refined 3D reconstruction obtained from the data collected. (a) Data collected without using a VPP on a K2 Summit detector. (b) Data collected using a VPP on a K2 Summit detector. (c) Data collected using a VPP on a Falcon III detector in electron counting (EC) mode. (d) Gold-standard FSC curves for the three 3D reconstructions with resolutions estimated at 0.143. (e) Difference in B-factors between the three datasets.

Figure 3.. Local resolution cryo-EM map.

(a) Local resolution map of the Falcon III + VPP model prior to refinement with signal subtracted particles as calculated with RELION. (b) Local resolution of the same model after refinement of signal subtracted particles (also calculated with RELION) (c) A_2AR complex displayed as putty cartoons, where B-factor of the coordinates relates to the thickness of the tube. (d) Fourier shell correlation of the refined model versus the map.

Figure 4.. Modelling quality of the A_2AR structure.

(a) Amino acid sequence of A_2AR used in the cryo-EM structure determination. Residues are coloured according to how they have been modelled: black, good density allows the side chain to be modelled; red, limited density for the side chain present and therefore the side chain has been truncated to Cβ; blue, no density observed and therefore the residue was not modelled. Regions highlighted in grey represent the transmembrane α-helices and amphipathic helix eight is highlighted in yellow. Cys residues involved in the formation of disulphide bonds are in bold. In the cryo-EM structure densities for the disulphide bonds Cys74-Cys146 and Cys77-Cys166 are observed. Densities corresponding to the disulphide bonds Cys71-Cys159 and Cys259-Cys262 are not observed in the cryo-EM data. The sequence of A_2AR is from residue 8–316, with the initial Ala residue at position seven being part of the linker between the N-terminal thioredoxin fusion and A_2AR. (b) Model of A_2AR showing the Cα positions of amino acid residues with poor density (spheres) and regions unmodelled (dotted lines).

Figure 5.. Comparison of map densities from the cryo-EM data and X-ray diffraction data.

The structure of the A_2AR–heterotrimeric G protein complex determined by cryo-EM is depicted as a cartoon. The four panels show regions of the structure and the associated density maps from the cryo-EM data and, where present, electron density (2Fo-Fc) from the X-ray structure of the A_2AR–mini-G_S (PDB code 5g53). Densities for the maps shown in the panels were sharpened using the following B factors (resolution of filtering in parentheses): β subunit and A_2AR, −170 Ų (3.7 Å); mini-G_S–A_2AR interface, −130 Ų (3.7 Å); NECA, −130 Ų (4.1 Å).

Figure 6.. Structure of A_2AR–heterotrimeric G_S.

(a) Superposition of A_2AR (pale green) coupled to mini-Gs (pale blue) with A_2AR (dark green) coupled to mini-G_S (dark blue), βγ (red) and Nb35 (yellow). (b) Superposition of NECA bound to A_2AR in the cryo-EM and X-ray structures after alignment of A_2AR (PyMol). (c) The position of His264 in the cryo-EM structure (dark green, density shown by black mesh), differs from its position in the X-ray structure (light green). No density is observed for the side chain of Glu169 in the cryo-EM structure, but when modelled it would be too far away to make a contact with His264. (d) The interface between ICL1 of A_2AR (dark green) and the β subunit (red) is depicted, with density shown as a black mesh.

Figure 7.. Comparison of A_2AR and β₂AR coupled to heterotrimeric G_S.

(a) A_2AR (dark green) and β₂AR (dark grey) were aligned using regions of the receptors predicted to be within the cytoplasmic leaflet of the lipid bilayer. The position of mini-G_S (pale green) coupled to A_2AR is compared to the position of the GTPase domain of the α subunit (pale grey) coupled to β₂AR. The βγ subunits and Nb35 have been omitted for clarity. (b) Transmembrane region H1 and ECL1 in A_2AR (dark green) extends closer to the β subunit (pale green), whereas β₂AR (dark grey) is too far away from the β subunit (pale grey) to make extensive contacts.