Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region.pdf (2.64 MB)
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Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region.

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journal contribution
posted on 25.09.2018, 09:05 by Hong Huo, Alistair G. Fryatt, Louise K. Farmer, Ralf Schmid, Richard J. Evans
ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to PPADS action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of PPADS). We hypothesized that cysteine substitutions of residues involved in PPADS binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered PPADS affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of PPADS binding. This binding site overlapped with the ATP-binding site, indicating that PPADS sterically blocks agonist access. Moreover, PPADS induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes PPADS sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.

Funding

This work was supported by British Heart Foundation Studentship FS/09/059/27972 (to L. F.) and project Grant PG/11/64/28772.

History

Citation

The Journal of Biological Chemistry, 2018, 293 (33), pp. 12820-12831

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

VoR (Version of Record)

Published in

The Journal of Biological Chemistry

Publisher

American Society for Biochemistry and Molecular Biology

issn

0021-9258

eissn

1083-351X

Copyright date

2018

Available date

25/09/2018

Publisher version

http://www.jbc.org/content/293/33/12820

Language

en