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Two disparate ligand-binding sites in the human P2Y1 receptor.

ABSTRACT: In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

SUBMITTER: Zhang D

PROVIDER: S-EPMC4408927 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Two disparate ligand-binding sites in the human P2Y1 receptor.

Zhang Dandan D Gao Zhan-Guo ZG Zhang Kaihua K Kiselev Evgeny E Crane Steven S Wang Jiang J Paoletta Silvia S Yi Cuiying C Ma Limin L Zhang Wenru W Han Gye Won GW Liu Hong H Cherezov Vadim V Katritch Vsevolod V Jiang Hualiang H Stevens Raymond C RC Jacobson Kenneth A KA Zhao Qiang Q Wu Beili B

Nature 20150330 7547

In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding ...[more]

PMID: 25822790

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Project description:We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1 receptor. Mutated receptors were expressed in COS-7 cells with an epitope tag that permitted confirmation of expression in the plasma membrane, and agonist-promoted inositol phosphate accumulation was assessed as a measure of receptor activity. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to be involved in ligand recognition were replaced with alanine and, in some cases, by other amino acids. The potent P2Y1 receptor agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing the R128A, R310A, and S314A mutant receptors, and a markedly reduced potency of 2-MeSATP was observed with the K280A and Q307A mutants. These results suggest that residues on the exofacial side of TM3 and TM7 are critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or maximal effect of 2-MeSATP with the S317A mutant receptor. Alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold reduction in potency compared with that observed with the wild-type receptor. These residues thus seem to subserve a less important modulatory role in ligand binding to the P2Y1 receptor. Because changes in the potency of 2-methylthio-ADP and 2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at these mutant receptors, the beta- and gamma-phosphates of the adenine nucleotides seem to be less important than the alpha-phosphate in ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in diphosphate or monophosphate potency. This result may be indicative of a greater role of these TM5 residues in gamma-phosphate recognition. Taken together, the results suggest that the adenosine and alpha-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.

Project description:The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Project description:BackgroundBark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods.ResultsWe annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding.ConclusionsThe emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

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Two disparate ligand-binding sites in the human P2Y1 receptor.

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Two disparate ligand-binding sites in the human P2Y1 receptor.

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