Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

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Science  13 Jul 2012:
Vol. 337, Issue 6091, pp. 232-236
DOI: 10.1126/science.1219218

GPCR Close-Up

Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232) used a protein-engineering strategy to produce a stabilized version of the human A2Aadenosine receptor (A2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na+ on ligand binding and how cholesterol may contribute to GPCR stabilization.


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 A2A adenosine receptor by replacing its third intracellular loop with apocytochrome b562RIL 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 Asp2.50. 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.

G protein–coupled receptors (GPCRs) encompass the largest and most diverse family of membrane proteins in eukaryotes, sharing a common architecture of seven-transmembrane (7-TM) α helices. GPCRs transduce a variety of signals across the cell membrane that regulate diverse biological and (patho)physiological processes and, hence, are favored drug targets. Ligand-dependent GPCR activation triggers dramatic conformational changes in the 7-TM helical bundle that are coupled to activation of G proteins and other downstream effectors. Over the past 4 years, breakthroughs in protein engineering and crystallography have yielded structures of 14 different GPCRs responding to diffusible ligands in different functional states (19), providing the basic structural framework for understanding ligand binding and activation mechanisms. However, despite the progress in GPCR stabilization for crystallographic studies, the resolution of GPCR structures has remained in the range of 2.2 to 3.5 Å (1). The function of GPCRs relies on a specific lipid environment and often strongly depends on the presence of cholesterol and ions (1012). Our understanding of the structural mechanism of such effects has been limited due to the absence of higher-resolution GPCR structures.

We replaced the third intracellular loop (ICL3) of the human A2A adenosine receptor (A2AAR) with a thermostabilized apocytochrome b562RIL (BRIL) (13, 14) and determined the crystal structure of this chimeric protein [referred to as A2AAR-BRIL-ΔC (15)] in complex with a high-affinity, subtype-selective antagonist, ZM241385, at 1.8 Å resolution (table S1). The high resolution enabled us to identify a comprehensive network of 57 internal water molecules, a highly conserved binding site for a sodium ion, three cholesterol molecules, and 23 lipid acyl chains bound to the receptor.

The ligand binding and functional characteristics of the A2AAR-BRIL-ΔC fusion protein were extensively characterized and compared with the A2AAR-WT (wild-type) and A2AAR-ΔC (C-terminal truncation) constructs. Antagonist [3H]ZM241385 radioligand-binding and agonist-displacement assays confirmed that the ligand recognition site of the A2AAR-BRIL-ΔC fusion protein is very similar to that of both the A2AAR-WT and A2AAR-ΔC constructs (fig. S1). The BRIL insertion did not have an impact on receptor expression and trafficking to the cell plasma membrane (fig. S2), but, as expected, the insertion prevented the receptor construct from activating available Gs proteins (fig. S3).

The high-resolution receptor structure is nearly identical to the original 2.6 Å resolution crystal structure of A2AAR-T4L-ΔC/ZM241385 [Protein Data Bank identification number (PDB ID) 3EML] (16), with an all-atom root mean square deviation of 0.45 Å over 81% of A2AAR (excluding terminal residues 1 to 9, 305 to 307, the distal part of ECL2 147 to 162, and residues 199 to 232 flanking ICL3). The conformations of the cytoplasmic ends of helices V and VI near the BRIL junction sites closely resemble the conformations in the A2AAR structure with unmodified ICL3 (13, 17), in contrast to a distorted conformation caused by the T4L fusion in A2AAR-T4L-ΔC/ZM241385 (16). A distal part of the extracellular loop 2 (ECL2), which was missing in all inactive-state structures of A2AAR, is fully resolved (fig. S4). Most importantly, the higher resolution revealed atomic details for the structurally conserved regions, including intricate networks of water molecules, sodium ion, lipids, and cholesterols.

The 1.8 Å–resolution structure of A2AAR-BRIL-ΔC/ZM241385 contains 177 structured waters, 57 of which occupy the interior of the 7-TM bundle. The interior waters form an almost-continuous channel extending from the ligand-binding site to the site of G protein interaction (Fig. 1A), which is composed of three bulky water-molecule clusters as well as several scattered waters. The channel has two narrow “bottlenecks” restricted by Trp2466.48 and Tyr2887.53 (18) to slightly less than the diameter of one water molecule (2.4 and 2.0 Å, respectively). Rearrangements of the receptor backbone and side chains upon activation disrupt the channel continuity in these sites (Fig. 1B), substantially reducing its volume.

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-ΔC/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.

The first of the three water clusters, the extracellular (EC) cluster, located inside the orthosteric ligand-binding pocket, plays a role in ligand binding and selectivity (Fig. 1C and fig. S5) (19). One of the waters in this cluster stabilizes the conformation of a nonproline kink in helix III by forming hydrogen bonds with both the main-chain carboxyl of Ile803.28 and the main-chain nitrogen of Val843.32. In the active-like state of agonist-bound A2AAR structures (20, 21), the kink in helix III straightens, thereby precluding water binding, which points to a role of water rearrangement at this location in A2AAR activation. This ligand-induced and water-mediated conformational change is accompanied by an overall activation-related shift of helix III (20).

The second, central water cluster includes a sodium ion and 10 water molecules that completely fill a cavity in the middle of the 7-TM bundle (Fig. 1D). The central cluster spans a distance of more than 13 Å between two functionally important and conserved residues, Trp2466.48 and Tyr2887.53. Three water molecules from this cluster were previously observed in the A2AAR-T4L-ΔC/ZM241385 complex (fig. S6A) (16), and positions of 4 of the 10 waters are similar to those found in bovine rhodopsin (PDB ID 1U19) (fig. S6B) (22), suggesting that such an internal network is common in class A GPCRs and could be important for their function (23, 24). Finally, the intracellular (IC) cluster containing ~20 water molecules is found near the conserved D[E]RY motif (D, Asp; E, Glu; R, Arg; Y, Tyr) (Fig. 1E), which is inferred in the stabilization of different functional receptor states and in interactions with G proteins and other effectors.

Evidence for the presence of a sodium ion in the central water cluster came from a strong spherical electron density with a distorted octahedral coordination and short distances to coordinating atoms in its neighborhood. Such geometry was poorly compatible with the presence of a water molecule. However, Na+ modeled in this density is coordinated by five oxygen atoms, including OD1 of Asp522.50 (2.45 Å), OG of Ser913.39 (2.54 Å), and three waters (2.20, 2.45, and 2.59 Å) (Fig. 2A and fig. S7), which is in excellent agreement with coordination and distances for Na+ in protein structures (25). Moreover, the presence of Na+ in the structure is consistent with conditions employed during purification (0.8 M Na+) and crystallization (~0.15 M Na+). Allosteric effects of Na+ on ligand binding, stability, and crystallization of A2AAR have been documented in previous studies (11, 16, 26). We confirmed the allosteric effects of physiological concentrations of Na+ on agonist and antagonist binding and thermal stability of A2AAR (Fig. 3 and table S2).

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-ΔC/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.

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-ΔC 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-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 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. ΔTm, shift in the melting temperature.

During the past 40 years, allosteric modulation by Na+ has been observed for many GPCRs and was linked to motifs in helix II, including the highly conserved Asp2.50 (27). Mutation of this residue has been the subject of many studies on a multitude of receptors; in the GPCRDB database (28), Asp2.50 is mentioned more than 100 times for dozens of receptors of human origin and from other species. Asp2.50 has not been mutated in the A2AAR, but in the closely homologous A1 and A3 adenosine receptors, the Na+ effect was largely abrogated when the residue was mutated to alanine or asparagine, respectively (29, 30). Despite this indirect evidence, the nature of Na+ interactions with GPCRs remained hypothetical (31, 32), and the sodium ion remained undetected in crystal structures of GPCRs solved at medium resolution.

In this high-resolution A2AAR structure, we were able to determine the precise location of Na+, as well as to resolve the complete network of water molecules around the sodium ion and conformations of all residues involved in direct or water-mediated coordination. The central water cluster harboring Na+ is surrounded by and engaged in hydrogen bonding with several highly conserved residues, including Asn241.50, Asp522.50, Ser913.39, Trp2466.48, Asn2807.45, Asn2847.49, and Tyr2887.53 (Fig. 2A). Structural alignment of these residues from known GPCR structures reveals a well-preserved site capable of binding Na+ along with several water molecules (Fig. 2, C and D). Small deviations were observed in bovine rhodopsin, in which Asn7.45 is substituted with a serine residue and Asn7.49 adopts a different rotamer, and in CXCR4 where Asn7.45 is replaced with a histidine. Interestingly, in many olfactory receptors, Ser3.39 is substituted with a glutamate, resulting in two acidic side chains pointing inside the central water cluster, which may favor binding of a divalent cation in this site.

The central water cluster with Na+ likely plays an important role in receptor activation. Comparison between the inactive and active-like states of A2AAR (PDB ID 3QAK) suggests that, upon activation, the inward movement of helix VII in this region collapses the pocket from 200 to 70 Å3 and shifts it toward helix VI (Fig. 2B). The resulting pocket in the active-like state can accommodate a maximum of three water molecules and does not provide sufficient coordination for Na+. This comparison suggests that high-affinity agonist binding and the presence of Na+ in this site are mutually exclusive, which is consistent with the observed negative allosteric effects of Na+ on binding of agonists (Fig. 3 and table S2) (16, 20).

The high-resolution structure also allows us to investigate off-target interactions of the diuretic and sodium-channel blocker amiloride with GPCRs that were first documented by Howard et al. (33) and Nunnari et al. (34); they demonstrated that amiloride and derivatives noncompetitively displaced a radiolabeled antagonist from the α2-adrenergic receptor by enhancing the radioligand’s dissociation rate. Moreover, Howard et al. (33) provided evidence that NaCl competed with amiloride, suggesting that Na+ and amiloride share an allosteric binding site. We docked amiloride into the central cluster cavity (fig. S8), with only minor changes in the conformations of the surrounding side chains; i.e., Trp2466.48. Multiple docking runs consistently led to an amiloride orientation in which its charged guanidinium group interacts with Asp522.50, whereas other polar groups form a network of hydrogen bonds with residues in the pocket, mimicking polar interactions in the Na+/water cluster. Successful docking of bulkier amiloride derivatives, such as 5-(N,N-hexamethylene)amiloride, required more substantial rearrangements of several side chains and possibly the main chain, which would potentially perturb the conformation of the deepest part of the orthosteric ligand-binding pocket. This result is consistent with the higher potencies of such derivatives in accelerating orthosteric antagonists’ off rates (11). This docking study also corroborates the results of our experiments (Fig. 3), in which amiloride reduced [3H]ZM241385 binding (probably due to the conformational changes in Trp2466.48), inhibited binding of the agonist [3H]NECA, and competed with Na+ ions for the same binding site.

The 1.8 Å structure includes 23 ordered lipid chains and three cholesterols per receptor. Together, they form an almost-complete lipid bilayer around each protein molecule, mediating crystal contacts (Fig. 4, A and B, and fig. S9). Lipids on the EC side have stronger electron densities and appear to be more ordered. All three cholesterols in this structure are bound to the EC half of the receptor and have low average B-factors (25 to 27 Å2) in comparison to other lipids (41 to 61 Å2). Two of these cholesterols (CLR1 and CLR3) are bound to symmetry-related receptors and mediate crystal-lattice packing by forming face-to-face interactions. The third cholesterol molecule (CLR2) does not participate in crystal contacts. Interestingly, CLR2 and CLR3 occupy hydrophobic grooves along helix VI and form extensive contacts with the aromatic ring of Phe2556.57, which is sandwiched between these cholesterol molecules (Fig. 4C and fig. S10). In the adenosine family of receptors, position 6.57 is conserved as Ile, Val, or Phe: hydrophobic residues that all could support the type of stacking interaction observed in this structure. In addition, CLR2 forms a hydrogen bond (2.7 Å) with the main-chain carboxyl of Ser263, and the hydroxyl of CLR3 has a polar interaction with sulfur of Cys259 (3.8 Å) in ECL3, the loop that is stabilized by the Cys259-Cys262 disulfide bond. Specific binding and conformational stabilization of this region of helix VI by cholesterols may play a functional role in A2AAR by fixing the position of Asn2536.55 in the ligand-binding pocket of the receptor (Fig. 4C). This key residue exists in all adenosine receptors and anchors the exocyclic amine of the ligand’s central core in both agonist and antagonist complexes (20). Such direct cholesterol binding to A2AAR is also consistent with observations that addition of cholesterol hemisuccinate (CHS) increases the thermostability of the receptor purified in detergent micelles (Fig. 3).

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-ΔC/ZM241385 complex are shown as sticks with light blue carbons. The superimposed active-like state A2AAR-T4L-ΔC/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.

Although most lipid chains form nonspecific hydrophobic contacts filling grooves on the protein surface, several interactions of lipids with A2AAR may be specific: for example, interactions between the lipids’ polar groups and the main-chain and side-chain polar groups of ECL1 and ECL3. In addition, one lipid (OLA) intercalates inside the TM bundle between helices I and VII, thereby protruding into the ligand-binding pocket (Fig. 4D). This lipid molecule apparently stabilizes the conformation of the first eight N-terminal residues of helix I, which do not make any direct contacts with the rest of the helical bundle.

It is intriguing to speculate that GPCRs are allosteric machines (3537), controlled in part by ions like sodium, membrane components such as lipids and cholesterol, and also water molecules. Although this concept of allostery exceeds its common definition, with increased-resolution eukaryotic membrane protein structures and complementary biophysical studies, it seems likely that we will start to observe the control of membrane proteins, not only by pharmacological ligands, but also by endogenous small molecules at specific binding sites. The small molecules mentioned here can dramatically affect a protein’s stability and function, which can have a pronounced effect on the physiological signaling of GPCRs in very important ways.

Supplementary Materials

Materials and Methods

Figs. S1 to S10

Tables S1 and S2

References (3957)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. The construct is based on the sequences of the wild-type human A2AAR and the thermostabilized apocytochrome b562 from E. coli (M7W, H102I, K106L), referred to as BRIL, including the following features: (i) Residues Ala1 to Leu106 of BRIL were inserted between Lys209 and Gly218 within the A2AAR ICL3 region. (ii) C-terminal residues 317 to 412 of A2AAR were truncated.
  3. Superscripts refer to the Ballesteros-Weinstein numbering in which 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.
  4. Acknowledgments: This work was supported by NIH Common Fund in Structural Biology grant P50 GM073197 for technology development, NIH/National Institute of General Medical Sciences (NIGMS) PSI:Biology grant U54 GM094618 for biological studies and structure production, NIGMS grant R01 GM089857 to V.C., a Dutch Research Council Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP grant (714.011.001: “The crystal structure of the adenosine A2A receptor: The follow-up”) to A.P.I., and an NWO Veni grant (11188) to L.H.H. We thank J. Velasquez for help on molecular biology; T. Trinh, K. Allin, and M. Chu for help on baculovirus expression; T. Mulder-Krieger and H. de Vries for their technical expertise in the biochemical characterization, G. van Westen for his educated comments on the crystal structure; M. Mileni for reviewing the final structure; J. P. Changeux for discussions on GPCR allostery and the potential for allosteric stabilizers; K. Kadyshevskaya for assistance with figure preparation; A. Walker for assistance with manuscript preparation; 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 NIGMS grant Y1-GM-1104. Coordinates and the structure factors have been deposited in the Protein Data Bank under the accession code (4EIY). R.C.S. is a founder and on the Board of Directors of Receptos, a GPCR structure-based drug discovery company. R.C.S., C.B.R., A.A.T., W.L., F.X., E.C., and V.K. are inventors on a patent applied for jointly by The Scripps Research Institute and Receptos on the use of fusion proteins to crystallize GPCRs.
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