Kasuya G et al. (2017), Structural insights into the nucleotide base sp...

XB-ART-55452

Sci Rep 2017 Mar 23;7:45208. doi: 10.1038/srep45208.

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Structural insights into the nucleotide base specificity of P2X receptors.

Kasuya G , Fujiwara Y , Tsukamoto H , Morinaga S , Ryu S , Touhara K , Ishitani R , Furutani Y , Hattori M , Nureki O .

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P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.

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Species referenced: Xenopus laevis
Genes referenced: atr p2rx4 tbl1x

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	Figure 1. Current responses of zebrafish P2X4 evoked by nucleoside triphosphates.(A–D) Representative currents of zebrafish P2X4 WT, evoked first by 100 μM ATP and then by (A) 1 mM ATP, (B) 1 mM CTP, (C) 1 mM GTP and (D) 1 mM UTP. Each nucleotide was applied for 10 sec. The holding potential was −70 mV.
	Figure 2. Overall comparisons of the CTP-bound and ATP-bound ΔP2X4-C structures.(A,B) The CTP-bound ΔP2X4-C structure viewed parallel to the membrane (A) and from the extracellular side (B). (C) The omit Fo − Fc map contoured at 4σ, showing the electron density of CTP. CTP is depicted by a stick model. (D,E) The ATP-bound ΔP2X4-C structure viewed parallel to the membrane (D) and from the extracellular side (E). (F) The omit Fo − Fc map contoured at 4σ, showing the electron density of ATP. ATP is depicted by a stick model.
	Figure 3. Close-up views of the CTP and ATP binding sites.(A–D) Close-up views of the CTP binding site in the CTP-bound ΔP2X4-C structure (A,B), and the ATP binding site in the ATP-bound ΔP2X4-C structure (C,D). Side chains of amino acid residues and nucleoside triphosphates are depicted by stick models. The molecule is colored according to the previously proposed dolphin-like model. Each dotted black line and number indicates a hydrogen bond and its length (<3.3 Å) respectively. (E) Sequence alignment around agonist binding site of P2X receptors. Amino acid sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and are shown using ESPript3 (http://espript.ibcp.fr/ESPript/ESPript/). For the sequence alignment, zebrafish P2X4 (zfP2X4, GI: 12656589) and the following P2X receptors were used: human (hP2X1, 4505545; hP2X2, 25092719; hP2X3, 28416925; hP2X4, 116242696; hP2X5, 209572778; hP2X6, 6469324; and hP2X7, 29294631), rat (rP2X1, 1352689; rP2X2, 18093098; rP2X3, 1030065; rP2X4, 1161345; rP2X5, 1279659; rP2X6, 1279661; and rP2X7, 1322005), Gulf Coast tick (amP2X, GI: 346469461), and blood fluke (smP2X, 51988420).
	Figure 4. Current responses of rWT, rT186S, rH140A and rH140R evoked by ATP and CTP.(A,B) Representative currents of rWT evoked by ATP (1–300 μM) (A) and CTP (10–10,000 μM) (B). (C,D) Representative currents of the rT186S mutant evoked by ATP (1–300 μM) (C) and CTP (10–10,000 μM) (D). (E,F) Representative currents of the rH140A mutant evoked by ATP (1–300 μM) (E) and CTP (10–10,000 μM) (F). (G,H) Representative currents of the rH140R mutant evoked by ATP (1–300 μM) (G) and CTP (10–10,000 μM) (H). (I–K) Concentration-response curves evoked by ATP and CTP in the rWT and the rT186S mutant (I), in the rWT and the rH140A mutant (J) and in the rWT and the rH140R mutant (K). Symbols are defined in the figure, and bars depict means ± SEM (n = 7–9).
	Figure 5. ATR-FTIR spectroscopic analysis of zfP2X4 for the ATP and CTP bindings.(A) The reduction of the ligand binding induced difference spectra in the ΔP2X4-C zfWT, zfT189S and zfT189V mutants around the P-O stretching region (1250–800 cm−1) after washing treatment for 15–30 min. (B) The residual nucleotides after the washing treatment estimated from the ATR-FTIR analysis in (A).
	Figure 6. Model for the nucleotide base specificity of P2X receptors.(A–D) Schematic representations of the interactions between zfP2X4 and ATP (A), CTP (B), GTP (C) or UTP (D). Residues involved in nucleotide base recognition and each nucleoside triphosphate are depicted by stick models. Black dashed lines and numbers indicate hydrogen bond interactions, and red crosses indicate non-complementary hydrogen bond partners, despite reasonable hydrogen bonding distances.

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Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed