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, 337 (6091), 232-6

Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions


Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

Wei Liu et al. Science.


Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A(2A) adenosine receptor by replacing its third intracellular loop with apocytochrome b(562)RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp(2.50). Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.


Fig. 1
Fig. 1. Distribution of ordered waters in A2AAR
In all panels, the antagonist-bound high-resolution structure is shown in cyan and the agonist-bound, active-like state structure (PDB ID 3QAK) is shown in yellow, waters are represented as red spheres, and salt bridges and hydrogen bonds as small green spheres. (A) Interior watersin A2AAR-BRIL-ΔC/ZM241385 structure form an almost continuous water channel (grey; calculated using the program Hollow (37)) containing three major water clusters. (B) The channel is disrupted in the active-like state structure (PDB ID 3QAK). (C) Close-up of the extracellular (EC) water cluster deep in the ligand binding pocket. The water molecule W15 shown as a large red semitransparent sphere stabilizes the kink in helix III. (D) Close-up of the central cluster, which includes waters and a sodium ion (blue transparent sphere). Water molecules W34 and W33 stabilizing the Pro-induced kinks in helices VI and VII are shown as large red semitransparent spheres. (E) Close-up of the intracellular (IC) cluster around the D[E]RY motif in helix III. Despite of close proximity Arg1023.50 and Glu2286.30 do not form an ionic interaction, instead both amino acids form hydrogen bonds with neighboring waters. An alternative rotamer of Glu2286.30, making a potential 3.0 Å contact with Arg1023.50 side chain, is shown in a grey stick representation.
Fig. 2
Fig. 2. Structural details of the Na+ allosteric site in the inactive and active-like state A2AAR
(A) Sodium ion (blue sphere) in the middle of the 7TM bundle coordinated by highly conserved Asp522.50, Ser913.39 and 3 water molecules. Receptor is shown as a ribbon, with residues lining the Na+ cavity shown as sticks and transparent spheres with carbon atoms colored cyan and oxygen atoms red. Water molecules in the cluster are shown as small red spheres, while the salt bridge between Na+ and Asp522.50 and hydrogen bonds are shown as green dotted lines. (B) The pocket collapses in the active-like state A2AAR-T4L-ΔC/UK432,097 structure, precluding Na+ binding at this site (hatched sphere designates the position of Na+ in the inactive structure). (C) Structural conservation of the allosteric pocket among solved GPCR structures. (A2AAR - cyan, CXCR4 - green, rhodopsin - magenta, all other - grey). (D) Sequence conservation of the pocket residues among all class A GPCRs (shown as a residue profile in the top row), and among the solved GPCR structures.
Figure 3
Figure 3. Modulation of A2AAR by sodium ions, amiloride and cholesterol
(A) [3H]ZM241385 or (B) [3H]NECA equilibrium binding to A2AAR-WT and A2AAR–BRIL-ΔC constructs transiently expressed on HEK293 cell membranes in the presence of buffer (control) or buffer supplemented with 150 mM NaCl, 100 μM amiloride, combinations of 100 μM amiloride and 150 mM NaCl, and 150 mM choline chloride. The figures represent data combined from three separate experiments performed in duplicate. Differences in specific binding were analyzed by a Student’s t-test. Significant differences were observed for the effect of modulators on control binding and are noted as follows: ** p < 0.01, *** p < 0.001. Significant differences were observed for the effect of NaCl on amiloride modulation and noted as follows: # p < 0.05, ## p < 0.01. There was no significant effect of choline chloride on [3H]ZM241385 or [3H]NECA binding, further proof that Na+ rather than Cl ions caused the effect of NaCl. (C) Shifts in thermostability of A2AAR-BRIL-ΔC construct purified in detergent micelles upon addition of 150 mM NaCl, 100 μM amiloride, combinations of 100 μM amiloride and 150 mM NaCl, 1 μM ZM241385, 1 μM ZM241385 and 150 mM NaCl, and 0.01% CHS. Experiments with ZM241385 have been repeated 6 times, with a standard deviation of less than 1 °C. The composition of control buffer was 25 mM Hepes pH 7.5, 0.05% DDM, 0.01% CHS for all samples except for the study of the effect of CHS, in which the control buffer was 25 mM Hepes pH 7.5, 0.05% DDM, 800 mM NaCl.
Fig. 4
Fig. 4. Lipid-receptor interactions
(A) Top view of A2AAR (cyan ribbon) including crystallographic neighbors (green: translational symmetry, magenta: rotational, yellow: antiparallel arrangement). Cholesterol molecules are shown as balls with yellow carbons, lipid molecules are shown as sticks with grey carbons. (B) Side view of A2AAR. (C) Potential stabilizing effect of two cholesterols, CLR2 and CLR3, on the conformation of helix VI. Side chains Phe2556.57 and Asn2536.55 of the A2AAR-BRIL-ΔC/ZM241385 complex are shown as sticks with cyan carbons. The superimposed active-like state A2AAR-T4L-ΔC/UK432,097 is shown as orange ribbon (helix VI only) and sticks (Asn2536.55 side chain and NECA scaffold of the UK432,097 agonist only). (D) Lipid molecule (OLA11, grey balls) is inserted in between helices I and VII inside the ligand-binding pocket.

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