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Structural basis of the multiple ligand binding mechanisms of the P2X1 receptor

Acta Pharmacologica Sinica volume 46, pages 2564–2573 (2025)Cite this article

Abstract

As important modulators of human purinergic signaling, P2X1 receptors form homotrimers to transport calcium, regulating multiple physiological processes, and are long regarded as promising therapeutic targets for male contraception and inflammation. However, the development of drugs that target the P2X1 receptor, such as the antagonist NF449, is greatly hindered by the unclear molecular mechanism of ligand binding modes and receptor activation. Here, we report the structures of the P2X1 receptor in complex with the endogenous agonist ATP or the competitive antagonist NF449. The P2X1 receptor displays distinct conformational features when bound to different types of compounds. Despite coupling to the agonist ATP, the receptor adopts a desensitized conformation that arrests the ions in the transmembrane (TM) domain, aligning with the nature of the high desensitization rates of the P2X1 receptor within the P2X family. Interestingly, the antagonist NF449 not only occupies the orthosteric pocket of ATP but also interacts with the dorsal fin, left flipper, and head domains, suggesting a unique binding mode to perform both orthosteric and allosteric mechanisms of NF449 inhibition. Intriguingly, a novel lipid binding site adjacent to the TM helices and lower body of P2X1, which is critical for receptor activation, is identified. Further functional assay results and structural alignments reveal the high conservation of this lipid binding site in P2X receptors, indicating important modulatory roles upon lipid binding. Taken together, these findings greatly increase our understanding of the ligand binding modes and multiple modulatory mechanisms of the P2X1 receptor and shed light on the further development of P2X1-selective antagonists.

Keywords: Structural biology; Ligand binding mode; Ion channel; Purinergic P2X1 receptor

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Fig. 1: Cryo-EM structures of ATP-P2X1 and NF449-P2X1 complexes.
Fig. 2: Channel characteristics of P2X1 in different states.
Fig. 3: Binding modes of ATP and NF449.
Fig. 4: Cholesterol binding mode in P2X1 structures.
Fig. 5: Model of the P2X receptor activation cycle.

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Data availability

The coordinates and cryo-EM density maps for the structures of the ATP-P2X1 and NF449-P2X1 complexes have been deposited in the PDB database with the identification codes 9LX5 and 9LXC, respectively, and in the Electron Microscopy Data Bank under accession codes EMD-63466 and EMD-63473, respectively.

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Acknowledgements

The cryo-EM studies were performed at the electron microscopy facility of the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences. We thank Yue Zhou from SIMM for cryo-EM data collection. This work was supported by the CAS Strategic Priority Research Program XDB37030100 (QZ).

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Author notes

  1. These authors contributed equally: Yu-ting Qiang, Peng-peng Wu

Authors and Affiliations

  1. School of Pharmacy, Guizhou Medical University, Guizhou, 550025, China

    Yu-ting Qiang & Qiang Zhao

  2. Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China

    Yu-ting Qiang, Xin Liu, Li Peng, Li-ke Zhao, Ya-ting Chen, Zhao-bing Gao, Qiang Zhao & Kun Chen

  3. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    Peng-peng Wu, Zhao-bing Gao, Qiang Zhao & Kun Chen

  4. University of Chinese Academy of Sciences, Beijing, 100049, China

    Peng-peng Wu, Zhao-bing Gao, Qiang Zhao & Kun Chen

  5. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China

    Xin Liu & Qiang Zhao

  6. School of Pharmacy, Zunyi Medical University, Zunyi, 563006, China

    Ya-ting Chen & Qiang Zhao

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Contributions

YTQ established and optimized the protein purification procedures, prepared protein samples for cryo-EM data collection, and helped with manuscript preparation. PPW performed the functional assays and helped with the data analysis. XL and YTC helped with plasmid cloning and protein preparation. LP expressed the proteins. LKZ helped with the protein purification and data analysis. ZBG planned and analyzed the experiments and helped with manuscript preparation. KC collected the cryo-EM data, performed the cryo-EM data processing and analysis, as well as model building and structure refinement, wrote the first version of the manuscript, and helped with the data analysis. QZ initiated the project, planned and analyzed the experiments, supervised the research, and finalized the manuscript with input from all coauthors.

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Correspondence to Qiang Zhao or Kun Chen.

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Qiang, Yt., Wu, Pp., Liu, X. et al. Structural basis of the multiple ligand binding mechanisms of the P2X1 receptor. Acta Pharmacol Sin 46, 2564–2573 (2025). https://doi.org/10.1038/s41401-025-01512-y

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