Computational protocol: Cysteine Scanning Mutagenesis (Residues Glu52–Gly96) of the Human P2X1 Receptor for ATP

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Protocol publication

[…] The trimeric assembly of the human P2X1 receptor (residues 33–352) was modeled in Modeler version 9.7 () based on the zebrafish P2X4 structure (Protein Data Bank code 3I5D) as template. Target and template share 44% sequence identity and 67% sequence similarity based on a BLAST alignment. Of the models generated, the best model was selected based on Modeler's scoring function and external validation via Whatcheck () and PROSA (). This model then served as the starting structure for ligand docking with ATP and BzATP. In preparation for ligand docking, atomic coordinates for ATP were extracted from the Protein Data Bank (entry 1HCK). Hydrogens were added in Hyperchem 8.0 (Hypercube Inc., Gainesville, FL) using the MM+ force field to generate two alternative protonation states, ATP3- and ATP2-, followed by steepest descent energy minimization until convergence. In a similar way, BzATP was built and optimized based on the energy-minimized ATP structure. Both ATP and BzATP were docked into the P2X1 homology model using the GOLD package (). The approximate location of the ATP binding site was set based on previous work () and defined as all residues within a 20-Å sphere centered at the Phe291 backbone nitrogen. The side chains of residues Lys68, Lys70, Arg292, and Lys309 were treated as flexible using the “flexible side chain-library” option in GOLD to better accommodate the triphosphate moiety of ATP. The resulting docking poses were ranked by their GOLDScores and inspected manually. All figures illustrating molecular modeling results were prepared in PyMOL (version 1.2r3; Schrödinger, LLC), and internal cavities were visualized using the Hollow tool (). […]

Pipeline specifications

Software tools WHAT_CHECK, PyMOL
Application Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Chemicals Adenosine Triphosphate, Cysteine, Suramin