Computational protocol: Dynamic role of the tether helix in PIP2-dependent gating of a G protein–gated potassium channel

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Protocol publication

[…] The crystal structure of the GIRK2 channel bound to a PIP2 head group was used for MD simulations (see supplemental Materials and methods). We used the Protein Data Bank (PDB): 4KFM structure for simulations, because this channel is in a preopen conformation (). We compared the 3SYA and 4KFM structures and determined there is a small RMSD (, ), with the largest movements in the LM loop and a 0.41 Å RMSD for the c-α in the PIP2 pocket, formed by residues 88–92 (from the slide helix), 192–203 (HBC gate and tether helix), and 62–64 (N-terminal b-loop). Four 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol 4,5 bisphosphate (18:0, 22:4 PIP2) molecules were aligned to the crystal structure of PIP2, one at each binding site. Gβγ subunits were not included in the calculations. The GIRK2-PIP2 system was embedded in a bilayer consisting of 202 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; 16:0, 18:1 PC) lipids using the CHARMM-GUI membrane builder (; ). The protein–lipid system was then solvated with TIP3P water molecules and 150 mM KCl. The CHARMM36 force field was employed (). Additionally, five potassium ions were placed in the ion conduction pathway, and four sodium ions were placed in the sodium-binding site as seen in the crystal structures. A second system was set up with the same conditions but with a 6′Y mutation generated by in silico mutagenesis using CHARMM-GUI. The two systems (WT and 6′Y) were subjected to stepwise decreased restraint equilibration used in CHARMM-GUI membrane builder (). The two systems (WT and 6′Y) were then equilibrated for 20 ns using positional restrain of 1,000 KJ/mol/nm2 on all Cα atoms of the protein before the 400-ns unrestrained production simulations. We then repeated the 400-ns production simulations using different initial velocities for both systems. Simulations were conducted using GROMACS 4.6 () with a 2 fs integration time step and visualized using VMD, Pymol (), or Discovery Visualizer 4.0 (Accelrys). […]

Pipeline specifications

Software tools CHARMM-GUI Membrane Builder, CHARMM, GROMACS, VMD, PyMOL
Applications Protein structure analysis, Membrane protein analysis
Diseases Nervous System Diseases
Chemicals Ethanol, Amino Acids, Potassium