Computational protocol: Ion access pathway to the transmembrane pore in P2X receptor channels

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Protocol publication

[…] The zfP2X4 crystal structure (Protein Data Bank accession no. 3H9V) was modeled from residue 32 to 361, with missing side chains built using CHARMM. The rP2X2 receptor and the human P2X5 (hP2X5) receptor homology models were created based on the zfP2X4 receptor crystal structure using MODELLER software (). The electrostatic free energy of ion interaction along the two pathways was determined by calculating ΔΔGint = ΔGion+channel−ΔGchannel−ΔGion, with each component obtained from a distinct numerical solution of the finite-difference Poisson-Boltzmann equation for the given system. This procedure ensures that the contribution from the static field (arising from the protein charges) and the reaction field (arising from the spatial variations in the dielectric constant) are both incorporated into the calculated free energy. The calculations were performed following the methods described in , using the PBEQ module in CHARMM (version 35a1) and the set of optimized atomic Born radii for amino acids (; ; ). The zfP2X4, rP2X2, and hP2X5 systems were aligned with the central pore along the z axis. For the finite-difference Poisson-Boltzmann calculations, each system contained ∼160 × 160 × 290 grid points with cell size of 1.0 Å focused to 0.5 Å. The membrane was defined as a 25-Å-thick slab centered at Z = 0 Å with dielectric constant εM = 2, whereas protein and solvent dielectric constants were set to εP = 2 and εS = 80, respectively. Ions were treated explicitly inside a box defined from −15 Å < X < 15 Å, −15 Å < Y < 15 Å, and −20 Å < Z < 90 Å, and Debye-Hückel ionic screening corresponding to 150 mM of salt was considered outside this region. All residues were set to their native protonation states, with histidines neutral. For the lateral fenestrations, ΔΔGint was calculated for accessible points, those positions >2 Å from a protein atom, within a 20-Å radius of Z = 25 Å. The artificial opening of the central pore was constructed by carrying out an adopted basis Newton-Raphson minimization with a cylindrical potential resistant along the central axis of the protein, successively increasing the radius by 0.1 Å to a final radius of 5 Å. […]

Pipeline specifications

Software tools CHARMM, MODELLER
Application Protein structure analysis
Organisms Danio rerio, Dipturus trachyderma, Rattus norvegicus
Chemicals Adenosine Triphosphate