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

The Journal of Biological Chemistry
Hong HuoRichard J Evans

Abstract

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 acce...Continue Reading

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Citations

Jul 18, 2020·Medicinal Research Reviews·Francesco CalzaferriAntonio G García
May 21, 2020·Frontiers in Cellular Neuroscience·Paola SotoNelson Patricio Barrera
Jun 26, 2020·Frontiers in Pharmacology·Romain LaraShaun McNulty
Sep 26, 2020·International Journal of Molecular Sciences·Francisco Andrés Peralta, J Pablo Huidobro-Toro
Nov 2, 2020·Biochemical Pharmacology·Kenneth A JacobsonChrista E Müller
Sep 6, 2020·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Wen-Jun ZhangZheng-Ming Zhu
Aug 10, 2020·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Wen-Jun ZhangZhengming Zhu
Jan 14, 2022·Proteins·Danqi Sheng, Motoyuki Hattori

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Methods Mentioned

BETA
X-ray

Software Mentioned

RosettaLigand
GraphPad Prism
GraphPad
ImageJ
cpptraj
Flexstation
pClamp
Modeler
PyMol

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