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Agonist-bound structure of the human P2Y12 receptor.

Zhang J, Zhang K, Gao ZG, Paoletta S, Zhang D, Han GW, Li T, Ma L, Zhang W, Müller CE, Yang H, Jiang H, Cherezov V, Katritch V, Jacobson KA, Stevens RC, Wu B, Zhao Q - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China [2].

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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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.
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Figure 5: 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.

Mentions: All three P2Y12R structures were determined using the same thermostabilizing construct5. The receptor conformations of the 2MeSADP and 2MeSATP bound complexes are very similar (Cα R.M.S.D. = 0.6 Å), and both ligands overlay well in the same binding pocket (R.M.S.D. of common atoms = 0.6 Å), with the γ-phosphate group of 2MeSATP extended towards the extracellular surface (Fig. 1, Extended Data Fig. 1). Since the two structures are similar, we will focus our discussions on the higher resolution P2Y12R-2MeSADP structure and will point out the specific differences of the 2MeSATP bound complex as needed.


Agonist-bound structure of the human P2Y12 receptor.

Zhang J, Zhang K, Gao ZG, Paoletta S, Zhang D, Han GW, Li T, Ma L, Zhang W, Müller CE, Yang H, Jiang H, Cherezov V, Katritch V, Jacobson KA, Stevens RC, Wu B, Zhao Q - Nature (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128917&req=5

Figure 5: 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.
Mentions: All three P2Y12R structures were determined using the same thermostabilizing construct5. The receptor conformations of the 2MeSADP and 2MeSATP bound complexes are very similar (Cα R.M.S.D. = 0.6 Å), and both ligands overlay well in the same binding pocket (R.M.S.D. of common atoms = 0.6 Å), with the γ-phosphate group of 2MeSATP extended towards the extracellular surface (Fig. 1, Extended Data Fig. 1). Since the two structures are similar, we will focus our discussions on the higher resolution P2Y12R-2MeSADP structure and will point out the specific differences of the 2MeSATP bound complex as needed.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China [2].

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.

Show MeSH
Related in: MedlinePlus