Structure of the human P2Y12 receptor in complex with an antithrombotic drug

Abstract

P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors1. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo2, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation3,4, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor)5,6 suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors7,8. Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.

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Figure 1: Overview of the P2Y12R–AZD1283 complex structure.
Figure 2: P2Y12R ligand-binding pocket for AZD1283.
Figure 3: Hypothetical binding modes of other ligands to P2Y12R obtained by docking simulations.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the PDB under accession number 4NTJ.

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Acknowledgements

This work was supported by National Basic Research Program of China grants 2012CB910400 and 2012CB518000 (B.W., Q.Z.), National Institutes of Health (NIH) grants R01 AI100604 (B.W., Q.Z.) and U54 GM094618 (V.C., V.K., R.C.S.; Target GPCR-87), National Science Foundation of China grants 31370729 and 31170683 (B.W., Q.Z.), the National Institute of General Medical Sciences Postdoctoral Research Associate program (E.K.) and the NIH National Institute of Diabetes and Digestive and Kidney Diseases Intramural Research Program (K.A.J.). The authors thank AstraZeneca for their gift of AZD1283, and thank S. Nylander, F. Giordanetto and H. van Giezen for careful review and scientific feedback on the manuscript, A. Walker for assistance with manuscript preparation, and C. Wang and D. Wacker for help on collection of X-ray diffraction data.

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Contributions

K.Z. optimized the construct, expressed and purified human P2Y12R–BRIL for crystallization, developed the purification procedure, performed crystallization trials and optimized crystallization conditions. J.Z. helped in construct and crystal optimization, and collected diffraction data. Z.-G.G. designed, performed and analysed ligand-binding and competition assays of wild-type and mutant P2Y12R. D.Z. helped in expression and purification. L.Z. designed and made constructs for baculoviral expression. G.W.H. solved and refined the structure. S.M.M. performed and analysed ligand-binding assays. S.P. performed and analysed docking assays. E.K. helped in ligand synthesis of P2Y12R. W.L. helped in crystal optimization. G.F. helped in crystallographic data collection. W.Z. developed the initial expression and purification protocol for P2Y12R. C.E.M. provided compounds and discussed results. H.Y. helped to design and analysed docking assays. H.J. oversaw design and validation of P2Y12R models. V.C. helped to design and optimize LCP crystallization trials, collected and processed crystallographic data and wrote the manuscript. V.K. performed and analysed molecular modelling simulations, and wrote the manuscript. K.A.J. oversaw, designed and analysed ligand-binding assays and docking, and assisted with manuscript preparation. R.C.S. oversaw expression, purification and crystallization, and structure analysis/interpretation of P2Y12R. B.W. and Q.Z. initiated the project, planned and analysed experiments, supervised the research and wrote the manuscript.

Corresponding author

Correspondence to Qiang Zhao.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Chemical structures of different P2Y12R ligands.

a, AZD1283. b, ADP. c, 2MeSADP. d, R-138727. e, Diadenosine tetraphosphate (Ap4A).

Extended Data Figure 2 Size-exclusion chromatography traces, crystals and overall structure of P2Y12R–AZD1283 complex.

a, aSEC traces of P2Y12R–BRIL (black) and P2Y12R(D294N)–BRIL (red) purified in complex with AZD1283. The samples are expressed and purified in parallel from roughly the same amount of cells. b, Crystals of P2Y12R–BRIL and AZD1283 complex. The size of crystals is roughly 150 × 5 × 5 μm. c, Crystals of P2Y12R(D294N)–BRIL and AZD1283 complex. The size of crystals is roughly 150 × 15 × 15 μm. d, Cartoon representation of P2Y12R(D294N)–BRIL. The P2Y12R is shown in pale green ribbons, BRIL is in wheat ribbons, AZD1283 is magenta carbons, and cholesterols and lipids are yellow carbons. e, The 2mFo − DFc electron density map of the ligand-binding pocket contoured at 1.2σ. f, The 2mFo − DFc electron density map of the DRY motif region contoured at 1.2σ.

Extended Data Figure 3 Dimeric receptor association and interactions with cholesterols in the crystals of P2Y12R.

a, Two P2Y12R molecules make contact with each other, as mediated by helix V and two molecules of cholesterol related by a two-fold axis. a, b, The detailed interactions of the cholesterol molecules on helices III and V are shown in surface (b) and in cartoon (c) representations. The cholesterol molecule is coloured in yellow carbons, and P2Y12R is shown in pale green (surface) or grey (cartoon). Residues within 4 Å of cholesterols are represented as green sticks. Hydrogen-bond interactions are indicated by dashed lines. As the interactions of the two cholesterols at the interface are identical, only one is revealed in detail. d, e, The binding site of cholesterol between helices I and VII is shown in surface (d) and in cartoon (e) representation. AZD1283 is shown in magenta carbons.

Extended Data Figure 4 Comparison of relative residue positions between helix III and helix VI of P2Y12R, PAR1 and β2AR.

a, P2Y12R. b, PAR1. c, β2AR. The receptors are shown in grey ribbon representation. The DR3.50Y(F) motif and corresponding 6.37 (or 6.34) positions are shown as sticks of green, slate and blue carbons, respectively. d, Superimposition of P2Y12R with other GPCR structures. P2Y12R (pale green), β2AR (PBD accession 2RH1; blue), A2AAR (PDB accession 3EML; orange), κ-OR (PDB accession 4DJH; pink), NTSR1 (PDB accession 4GRV; yellow) and PAR1 (PDB accession 3VW7; slate) are superimposed and shown as ribbons. Transmembrane helices I, II and VII overlay relatively well, whereas the position of helix VI is substantially different in P2Y12R. e, Comparison of W(F)6.48 positions in P2Y12R (pale green), β2AR (blue), A2AAR (orange), κ-OR (pink) and PAR1 (light blue). For receptors other than P2Y12R, only helix VI is shown, and the residues at position 6.48 are shown as sticks. f, The comparison of ligand-binding sites of GPCRs from α (β2AR and A2AAR), β (NTSR1), γ (CXCR4; PDB accession 3OE0) and δ (P2Y12R and PAR1) subgroups. The structure of P2Y12R is shown in grey cartoon representation and AZD1283 is shown as magenta sticks. The ligands from other receptors (carazolol, cyan; ZM241385, green; neurotensin, red cartoon; IT1t, purple; vorapaxar, yellow) are placed at their corresponding positions in the 7TM bundle.

Extended Data Figure 5 The conserved helix III–ECL2 disulphide bond might be dynamic in AZD1283-bound P2Y12R.

a, b, The electron density of helix V (a) and helix III (b). Electron density is represented by a 2mFo − DFc map countered at 1.2σ. The K179 and C973.25 side chains are indicated with black arrows. cf, Effects of cysteine mutations on P2Y12R–BRIL. As the stability of wild-type P2Y12R is very poor, all the constructs here contain fusion protein and D294N mutation as described in the crystallization of the receptor. c, Comparison of aSEC traces of P2Y12R apo and P2Y12R, C97A, C175A in complex with AZD1283. The samples were treated with iodoacetamide (IA) before extraction. d, Comparison of aSEC traces of P2Y12R, C17A, C270A and C17A/C270A in complex with AZD1283. e, aSEC curves of purified P2Y12R (red), P2Y12R C97A (blue) and P2Y12R C175A (purple) with R-138727, the active metabolite of prasugrel, which binds irreversibly to P2Y12R by interacting with its cysteine residue(s). aSEC curve of apo P2Y12R is shown in black. It is obvious that treatment of R-138727 greatly improves the homogeneity of P2Y12R, which is not affected by the C175A mutation. However, the C97A mutation almost completely abolishes the effect of R-138727, indicating that C973.25 is the binding site of this compound. f, The melting curves of P2Y12R, C97A, C175A binding with AZD1283 and P2Y12R in the apo form.

Extended Data Figure 6 The hypothetical binding modes of 2MeSADP to antagonist-bound state P2Y12R.

a, b, An electrostatics surface representation (a) and a cartoon representation (b) of the hypothetical docking model of P2Y12R with bound 2MeSADP in pocket 2. The agonist 2MeSADP is shown as sticks (deep blue carbons). The side chains of residues that are involved in the binding of 2MeSADP are also labelled and shown as sticks (yellow carbons). c, d, An electrostatics surface representation (c) and a cartoon representation (d) of the hypothetical docking model of P2Y12R with bound 2MeSADP in pocket 1.

Extended Data Figure 7 Representative saturation curves of [3H]2MeSADP-specific binding to wild-type P2Y12R and various mutant receptors at a concentration range of 0.4–46 nM.

The calculated Kd values from 3–6 independent experiments are listed in Extended Data Table 3. Non-specific binding was determined using 10 µM AZD1283.

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Binding affinities for different P2Y12R constructs
Extended Data Table 3 [3H]2MeSADP binding affinities for different P2Y12R mutants

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Zhang, K., Zhang, J., Gao, Z. et al. Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature 509, 115–118 (2014). https://doi.org/10.1038/nature13083

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