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. 2014 May 1;509(7498):119-22.
doi: 10.1038/nature13288.

Agonist-bound structure of the human P2Y12 receptor

Jin Zhang  1 , Kaihua Zhang  1 , Zhan-Guo Gao  2 , Silvia Paoletta  2 , Dandan Zhang  3 , Gye Won Han  4 , Tingting Li  3 , Limin Ma  3 , Wenru Zhang  3 , Christa E Müller  5 , Huaiyu Yang  6 , Hualiang Jiang  6 , Vadim Cherezov  4 , Vsevolod Katritch  4 , Kenneth A Jacobson  2 , Raymond C Stevens  7 , Beili Wu  3 , Qiang Zhao  3
Affiliations

Agonist-bound structure of the human P2Y12 receptor

Jin Zhang et al. Nature. .

Abstract

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5'-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5'-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283), reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.

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Figures

Fig. 1
Fig. 1
Overall structure of the P2Y12R-2MeSADP and P2Y12R-2MeSATP complexes. (a) Side view of P2Y12R-2MeSADP complex structure. The receptor is colored cyan and shown in cartoon representation. The ligand 2MeSADP is shown in sphere representation with orange carbons. The disulfide bonds are shown as yellow sticks; extracellular and intracellular boundaries are shown as dashed lines. (b) Side view of P2Y12R-2MeSATP complex structure. The receptor is colored violet and shown in cartoon representation. The ligand 2MeSATP is shown in sphere representation with gray carbons. (c) Semitransparent surface presentation of the receptor shows the lid formed by ECL2, ECL3 and N-terminus on top of 2MeSADP. (d) Comparison of P2Y12R-2MeSADP with P2Y12R-2MeSATP complex. The ligands are shown as sticks, and phosphorus in 2MeSATP is colored in green to be distinguished from 2MeSADP.
Fig. 2
Fig. 2
Comparison of the P2Y12R-2MeSADP (agonist) and P2Y12R-AZD1283 (antagonist) complexes. (a) The P2Y12R-2MeSADP complex (receptor: cyan cartoon and ligand: sticks of orange carbons) and P2Y12R-AZD1283 complex (receptor: light orange cartoon and ligand: sticks of green carbons) are shown in side view. Movement of the extracellular tips of helices VI and VII toward the center of the 7TM domain is shown by arrows. The extracellular (b) and intracellular (c) views of the comparison are also shown.
Fig. 3
Fig. 3
P2Y12R ligand binding pocket for 2MeSADP. (a) The receptor is shown in cyan cartoon representation. The ligand 2MeSADP (orange carbons) and receptor residues (slate carbons) involved in ligand binding are shown in stick representation. Other elements are colored as follows: oxygen, red; nitrogen, dark blue; sulfur, yellow; phosphorus, orange. The water molecules interacting with 2MeSADP are shown as red spheres. (b) Comparison of the 2MeSADP and AZD1283 binding poses in the overlaid P2Y12R complexes. Color scheme as in Fig. 2. (c) Summary of receptor interactions of 2MeSADP. Hydrogen bonds are displayed as blue dashed lines and the salt bridges as red dashed lines. The π-π interaction between 2MeSADP and Y1053.33 is indicated as green dashed lines. The NHα and COα indicate the main chain amine and carbonyl groups of the corresponding residue.
Fig. 4
Fig. 4
Schematic illustration of conformational changes in P2Y12R extracellular region. (a) Unliganded (apo) state of P2Y12R with open entrance to the pocket and partially disordered lid. The large number of uncompensated electrostatic charges of the side chains forming the pocket (R19Nterm, K802.60, R933.21, K173ECL2, K174 ECL2, H1875.36, R2566.55 and K2807.35) disfavor formation of the closed state. (b) The closed state is stabilized by binding of nucleotide agonist (e.g. ADP, 2MeSADP). (c) A similar conformation with “lid” closure occurs in 2MeSATP structure and for various docked N6 unsubstituted nucleoside triphosphate and triphosphate-mimetic ligands. (d) A helical reorganization is proposed for some N6 substituted nucleoside triphosphate and triphosphate mimetic ligands, especially with bulky N6 substituents. (e) Binding of non-nucleotide antagonist AZD1283 blocks inward movement of helices VI and VII and prevents “lid” closure.
Extended Data Figure 1
Extended Data Figure 1
Crystals and electron density of nucleotides for P2Y12R-2MeSADP and P2Y12R-2MeSATP complexes. (a) Crystals of the P2Y12R-2MeSADP complex. The size of the crystals is roughly 80×50×5μm; (b) Crystals of the P2Y12R-2MeSATP complex. The size of the crystals is roughly 30×30×5μm; (c) The 2mFo-DFc map for the 2MeSADP contoured at 1σ; (d) The 2mFo-DFc map for the P2Y12R-2MeSATP contoured at 1σ. The relatively high B-factor of the γ-phosphate group (98 Å2) compared with β-phosphate and surrounding protein atoms (∼75 Å2), and the propensity of 2MeSATP to hydrolyze to 2MeSADP suggest partial occupancy for the β -phosphate group. However, given the differences in crystal forms and packing, as well as the clear density of the γ-phosphate group, the P2Y12R-2MeSATP complex structure should provide relevant information about 2MeSATP binding.
Extended Data Figure 2
Extended Data Figure 2
Crystal packing of P2Y12R-2MeSADP, P2Y12R-2MeSATP and P2Y12R-AZD1283 complexes. (a) Overall structure of the P2Y12R-2MeSADP complex, P2Y12R and BRIL are shown in cyan and blue, respectively. (b and c) Crystal packing of P2Y12R-2MeSADP complex at two different views. (d) Overall structure of the P2Y12R-2MeSATP complex, P2Y12R is shown in pink. (e and f) Crystal packing of P2Y12R-2MeSATP complex at two different views. (g) Overall structure of the P2Y12R-AZD1283 complex, P2Y12R is shown in orange. (h and i) Crystal packing of P2Y12R-AZD1283 complex at two different views.
Extended Data Figure 3
Extended Data Figure 3
Comparison of antagonist- (orange) and agonist- (greencyan) bound P2Y12R structures with PAR1 structure (yellow). (a) Side view of the three structures. The receptor structures are shown as cylindrical helices, and AZD1283 and 2MeSADP are shown as sticks with green carbons and wheat carbons respectively. (b) Comparison view from the extracellular side. (c) Comparison view from the intracellular side.
Extended Data Figure 4
Extended Data Figure 4
The distortion of helix III by the disulfide bond. (a) Comparison of P2Y12R-AZD1283 (orange) and P2Y12R-2MeSADP (greencyan). (b) Corresponding positions of residues around C973.25 in P2Y12R-AZD1283 (orange) and P2Y12R-2MeSADP (greencyan).
Extended Data Figure 5
Extended Data Figure 5
Comparison of pocket 1 (a-c) and pocket 2 (d-f) of P2Y12R structures with different ligands. (a and d) The P2Y12R-2MeSADP structure. (b and e) The P2Y12R-2MeSATP structure. (c and f) The P2Y12R-AZD1283 structure. The 2MeSADP, 2MeSATP and AZD1283 ligands are shown in sticks with wheat, gray and green carbons, respectively.
Extended Data Figure 6
Extended Data Figure 6
Functional properties of different ligands at P2Y12R. Data (mean ±SEM) were determined in triplicate. (a) Parallel right shifts induced by antagonist AZD1283 (AZD) of the activation curves by agonist 2MeSADP in inhibition of cAMP production in P2Y12R/CHO cells. (b) Parallel right shifts induced by antagonist Ticagrelor (Tica) of the activation curves by agonist 2MeSADP in inhibition of cAMP production in P2Y12R/CHO cells. The pKB values of AZD and Tica are 8.17 ± 0.45 and 7.70 ± 0.18, respectively. (c) Partial agonist effects of AR-C66096 (ARC) in inhibition of cAMP production in P2Y12R/CHO cells. The EC50 value of AR-C66096 was determined to be 34.9 ± 2.9 nM, and its Emax 41.9 ±3.6% compared with 2MeSADP as 100%. A final concentration of 10 μM forskolin was used in the experiment. DMSO was used as a solvent for the stock solution of forskolin, AZD1283 and Ticagrelor. The stock solution of AR-C66096 was made with water.
Extended Data Figure 7
Extended Data Figure 7
Docking models of different nucleotide analogs to the structure of P2Y12R. (a) The crystal structure of P2Y12R-2MeSADP complex. (b) Docking of 2MeSADP to the P2Y12R structure. (c) Docking of ADP to the P2Y12R structure. (d) The crystal structure of P2Y12R-2MeSATP complex. (e) Docking of 2MeSATP to the P2Y12R structure. (f) Docking of ATP to the P2Y12R structure. (g) Docking of AR-C66096 to the P2Y12R structure. (h) Docking of AR-C67085 to the P2Y12R structure. (i) Docking of AR-C69931MX (Cangrelor) to the P2Y12R structure. 2MeSADP and 2MeSATP poses from corresponding crystal structures are shown in stick with orange and gray carbons respectively. Docking was performed to the conformation of P2Y12R found in the 2MeSADP-bound structure, and the docked ligands are shown in sticks with purple carbons. AR-C66096 and AR-C67085 show the same interactions observed in the 2MeSADP complex. In addition, the C2-propylthio substituent of AR-C66096 and AR-C67085 is located in a hydrophobic pocket in proximity to helix IV surrounded by F1063.34, Y1093.37, M1524.53 and L1554.56. The γ-phosphonate group is directed towards helix III and interacts with K802.60 and R933.21. The C2 substituent and the γ-phosphonate group of AR-C69931MX show similar orientation as observed in the docking pose of AR-C66096 and AR-C67085. The N6 substituent is directed towards helix VI in proximity to Y1093.37, Q1955.44, F2526.51, H2536.52 and R256 6.55.
Extended Data Figure 8
Extended Data Figure 8
Ligands used in the docking studies. The chemical structures of parts of ligands that are discussed and used in the docking studies are shown. Ticagrelor and AR-C78511 could not be docked in a conformation similar to 2MeSADP because the presence of their bulky N6 substituents would cause a steric clash with helices V and VI. AR-C78511 was previously shown to lack partial agonist properties.
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References

    1. Chang H, et al. Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP-induced platelet activation. J Thromb Haemost. 2012;10:2573–2580. - PMC - PubMed
    1. Chang H, et al. Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors. Thromb Res. 2010;125:159–165. - PMC - PubMed
    1. Schmidt P, et al. Identification of determinants required for agonistic and inverse agonistic ligand properties at the ADP receptor P2Y12. Mol Pharmacol. 2013;83:256–266. - PMC - PubMed
    1. Srinivasan S, et al. The P2Y12 antagonists, 2-methylthioadenosine 5′-monophosphate triethylammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels. J Biol Chem. 2009;284:16108–16117. - PMC - PubMed
    1. Zhang K, et al. Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature. 2014 - PMC - PubMed

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