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Science 337 (6091): 232-236

Copyright © 2012 by the American Association for the Advancement of Science

Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

Wei Liu1,*, Eugene Chun1,*, Aaron A. Thompson1,*, Pavel Chubukov1, Fei Xu1, Vsevolod Katritch1, Gye Won Han1, Christopher B. Roth2, Laura H. Heitman3, Adriaan P. IJzerman3, Vadim Cherezov1,{dagger}, and Raymond C. Stevens1,{dagger}

1 Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
2 Receptos, San Diego, CA 92121, USA.
3 Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Post Office Box 9502, 2300RA Leiden, Netherlands.

Figure 1
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Fig. 1. Distribution of ordered waters in A2AAR. In all panels, the antagonist-bound, high-resolution structure is shown in light blue, the agonist-bound structure in the active-like state (PDB ID 3QAK) is shown in yellow, waters are represented as red spheres, and salt bridges and hydrogen bonds are depicted as small green spheres. (A) Interior water molecules in the A2AAR-BRIL-{Delta}C/ZM241385 structure form an almost-continuous water channel [gray; calculated using the program HOLLOW (38)] containing three major water clusters. (B) The channel is disrupted in the structure in the active-like state (PDB ID 3QAK). (C) Close-up view of the 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. I, Ile; A, Ala; C, Cys; V, Val; F, Phe. (D) Close-up view of the central cluster, which includes waters and a sodium ion (blue transparent sphere). Water molecules W34 and W33 stabilizing the proline-induced kinks in helices VI and VII are shown as large red semitransparent spheres. L, Leu; P, Pro; N, Asn; S, Ser. (E) Close-up view of the IC cluster around the D[E]RY motif in helix III. Despite their 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, which potentially makes a 3.0 Å contact with Arg1023.50 side chain, is shown in a gray stick representation. W246 is Trp246, but W15, W33, and W34 are waters 15, 33, and 34.


Figure 2
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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 7-TM bundle coordinated by highly conserved Asp522.50, Ser913.39, and three water molecules. The receptor is shown as a ribbon, and residues lining the Na+ cavity are shown as sticks. Transparent spheres with carbon atoms are colored light blue; oxygen atoms are transparent and red. Water molecules in the cluster are shown as small red spheres, whereas the salt bridge between Na+ and Asp522.50 and hydrogen bonds are shown as green dotted lines. T, Thr. (B) The pocket collapses in the active-like state A2AAR-T4L-{Delta}C/UK432,097 structure, precluding Na+ binding at this site (the hatched sphere designates the position of Na+ in the inactive structure). (C) Structural conservation of the allosteric pocket among solved GPCR structures. (A2AAR, light blue; CXCR4, green; rhodopsin, purple; all other, gray). (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. H, His.


Figure 3
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Fig. 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-{Delta}C constructs transiently expressed on human embryonic kidney 293 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 (**P < 0.01, ***P < 0.001), as well as for the effect of NaCl on amiloride modulation (#P < 0.05, ##P < 0.01). There was no significant effect of choline chloride on [3H]ZM241385 or [3H]NECA binding, which is further proof that Na+ rather than Cl ions caused the effect of NaCl. Error bars indicate SEM of three separate experiments. (C) Shifts in thermostability of A2AAR-BRIL-{Delta}C 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 were repeated six times, with a SD of less than 1°C. The composition of the control buffer was 25 mM Hepes pH 7.5, 0.05% n-dodecyl β-D-maltoside (DDM), and 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, and 800 mM NaCl. {Delta}Tm, shift in the melting temperature.


Figure 4
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Fig. 4. Lipid-receptor interactions. (A) Top view of A2AAR (light blue ribbon) including crystallographic neighbors (green, translational symmetry; purple, rotational; yellow, antiparallel arrangement). Cholesterol molecules are shown as balls with yellow carbons; lipid molecules are shown as sticks with gray 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-{Delta}C/ZM241385 complex are shown as sticks with light blue carbons. The superimposed active-like state A2AAR-T4L-{Delta}C/UK432,097 is shown as an orange ribbon (helix VI only) and sticks (Asn2536.55 side chain and NECA scaffold of the UK432,097 agonist only). (D) A lipid molecule (OLA, gray balls) is inserted in between helices I and VII inside the ligand-binding pocket.


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