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P2Y1‐like nucleotide receptors—Structures, molecular modeling, mutagenesis, and oligomerization

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Alexander Neumann, Christa E. Müller, Vigneshwaran Namasivayam

Published Online: Mar 03 2020

DOI: 10.1002/wcms.1464

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Abstract The P2Y receptors (P2YRs) are G protein‐coupled receptors (GPCRs) consisting of eight members, subdivided into two groups, P2Y1‐ and P2Y12‐like receptor subtypes. They are activated by extracellular nucleotides and represent current (P2Y2, P2Y12) or potential future drug targets. The chemical nature of the highly polar endogenous agonists represents a challenge in the discovery and design of potent and bioavailable compounds. A number of mutants and several homology models of P2YR subtypes have been created and updated on the basis of the recently published X‐ray crystal structures of the human P2Y1 and P2Y12Rs. The models were used for prediction of the binding sites of agonists and antagonists, and mutants were constructed for confirmation. Pharmacological data on mutants published for the P2Y1‐like receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11R) were evaluated to analyze the role of specific amino acids and that of corresponding amino acid residues in related P2Y receptor subtypes. In several P2YR subtypes, an ionic lock between extracellular loop 2 and transmembrane region VII was postulated to be essential for agonist‐induced receptor activation. Mutagenesis and homology modeling data suggest that the nucleotide antagonist (1′R,2′S,4′S,5′S)‐4‐(2‐iodo‐6‐methylaminopurin‐9‐yl)‐1‐[(phosphato)methyl]‐2‐(phosphato)bicyclo[3.1.0]hexane (MRS2500), which was co‐crystallized with the human P2Y1R, binds differently from agonistic nucleotides to a site partly overlapping with that of orthosteric agonists. Hetero‐oligomerization of P2YRs with other P2YR subtypes or other GPCRs may allosterically modulate receptor‐ligand interactions and/or G protein coupling. The collected information will contribute to the understanding of the architecture of P2Y1‐like nucleotide receptors and will consequently be useful for the design of novel agonists and antagonists. This article is categorized under: Molecular and Statistical Mechanics > Free Energy Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Interactions Software > Molecular Modeling

Schematic presentation of P2YRs embedded in a phospholipid bilayer and their preferred endogenous agonists. A phylogenetic tree shows the degree of their relationship. The endogenous agonists activating the respective receptor at physiologically relevant concentrations and the G protein coupling are shown at the top. Phylogenetic analysis was generated using Clustal Omega

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Oligomerization of P2Y receptors. (a) Oligomerization of P2Y₁R with P2Y₁₁R and the resulting impact on the pharmacological profile of the P2Y₁₁R. (b) Dimerization of P2Y₁R and P2Y₁₂R modulates the responses of the receptors toward K_2P ion channels when coexpressed in the same cell line. The profile of the dimers is similar to the pharmacological profile of the P2Y₁₂R, while the G protein‐coupling seems to be comparable to that of the P2Y₁R. Oligomerization of the adenosine A₁ receptor with the P2Y₁R (c) and the P2Y₂R (d). The oligomeric A₁AR/P2YR exhibits a preference for the corresponding P2YR agonists, whereas the potency of A₁AR agonist and agonists is reduced

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Overlay of the agonist‐bound X‐ray crystal structures of the human P2Y₁₂R, the nucleotide antagonist‐bound X‐ray crystal structure of human P2Y₁R, as well as the proposed binding mode of UTP in complex with the homology model of human P2Y₂R. The P2Y₁₂R complex with the agonist 2‐MeSADP is colored in yellow, the P2Y₁₂R complex with agonist 2‐MeSATP is colored in red, the P2Y₁R complex with antagonist MRS2500 (2.2) is colored in blue, the homology model P2Y₂R complex with agonist UTP is colored in green. The ligands are shown as stick models and the carbon atoms are colored respective to their receptor. Nitrogen atoms are colored in blue, oxygen in red, phosphorus in orange, sulfur in yellow. The β‐ and γ‐phosphate groups of the UTP bind close to a region occupied by the 5′‐phosphate group of the P2Y₁ antagonist MRS2500 (2.2)

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Overlay of RB‐2 (2.9) and the smaller anthraquinone derivative PSB‐16133 (2.14) in complex with the homology model of the human P2Y₄R. The receptor is presented in cartoon style. The helices are colored in red, the extracellular loops in green, and the β‐sheet of ECL2 in yellow. The antagonists are presented as stick models. RB‐2 is colored in blue, the smaller, most potent anthraquinone derivative PSB‐16133 is colored in orange

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Comparison of residues between ECL2 and TMVII which might play a role in receptor activation. P2Y₁R data was taken from the X‐ray crystal structure (PDB‐ID: 4XNW), P2Y₂R and P2Y₄R data from published homology models based on the P2Y₁R structure. The ionic lock could be induced by rotamer library selection. Ionic contact is presented by dashed lines

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Cartoon‐style presentation of published X‐ray crystal structures of P2YRs and their respective PDB‐ID. On the left side, both of the antagonist‐bound structures of the human P2Y₁R are shown in complex with 2.02 (MRS2500, PDB‐ID.: 4XNW) in light blue, and with 2.05 (BPTU, PDB‐ID: 4XNV) in violet blue. On the right side, the three available structures of the P2Y₁₂R are shown: in red the complex with the agonist 2‐MeSADP (2‐MeSADP, PDB‐ID: 4PXZ), in yellow the complex with the agonist 2‐MeSATP (1.07, PDB‐ID: 4PY0), and in orange the complex with a competitive antagonist 3.05 (AZD1283, PDB‐ID: 4NTJ). The receptors are presented as cartoon models, the co‐crystallized atoms of the ligands are presented as spheres: carbon atoms are colored in white, nitrogen atoms in blue, oxygen atoms in red, phosphorus atoms in orange, sulfur atoms in yellow, iodine atoms in purple

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Schematic diagram of available mutagenesis data of the human P2Y₁R, and effects of mutations on agonist and antagonist potencies. High impact was defined as at least 10‐fold change in the potency of the agonist. Changes below a 10‐fold difference in potency of the agonist were defined as low impact

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Schematic presentation of mutated residues in the human P2Y₂R homology model based on the human P2Y₁R X‐ray crystal structure. Transmembrane regions are presented as cylinders. Mutations of residues highlighted in red led to a large decrease in potency or complete abolishment of the receptor's ability to be activated by agonists. Mutation of green colored residues had a moderate impact on agonist potency. Purple‐colored mutants led to a decrease in potency only for ATP

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Schematic tree diagram of available mutagenesis data for agonist and antagonist potencies of the human P2Y₂R

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Schematic presentation of mutated residues depicted in the X‐ray crystal structure of the human P2Y₁R (PDB: 4XNW). Transmembrane regions are presented as cylinders. Roman numbers indicate the transmembrane regions. Mutations of residues highlighted in red led to a large decrease in potency or complete abolishment of the receptor's ability to be activated by agonists. Mutation of green colored residues had no or low impact on agonist potency. Blue highlighted residues were shown to participate in the binding of different antagonists

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Sequence alignment of the human P2Y₁‐like receptor subtypes. Residues important for agonist‐induced receptor activation predicted by MD simulations are highlighted in blue. Amino acid residues mutated in more than one receptor subtype are highlighted in red. Multiple sequence alignment was generated using Clustal Omega. MD, molecular dynamics

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Structures of selected P2Y₁₂R antagonists and A₁AR agonists and antagonists

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Structures of selected P2YR antagonists

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Structures of selected P2YR antagonists

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Structures of selected (proposed) P2Y receptor agonists

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