Computational protocol: Identification of the Conformational transition pathway in PIP2 Opening Kir Channels

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Protocol publication

[…] Homology models of the full-length mouse Kir2.1 channel were performed using the SWISS-MODEL server. The target sequences were taken from Genbank (http://www.ncbi.nlm.nih.gov/Genbank/). The template structures were the chicken Kir2.2 (PDB code: 3JYC and 3SPI), and the mouse Kir3.2 (PDB code: 3SYQ). These homology models were based on chain A of the template structures, in which the two missing loops, one that connects the N-terminal beta-strand and the slide helix, and one that connects the two transmembrane alpha-helices, were completed. All the models were evaluated with QMEAN (The QMEAN4 score is a composite score consisting of a linear combination of 4 statistical potential terms (estimated model reliability between 0–1). The pseudo-energies of the contributing terms are given their Z-scores with respect to scores obtained for high-resolution experimental structures of similar size solved by X-ray crystallography. The members of the inwardly rectifying K+ channel family exhibit high degree of sequence similarity. Due to high sequence homology (about 78%, 77% and 53% sequence identity to chicken Kir2.2 and mouse Kir3.2 respectively), the QMEAN4 score was 0.79, 0.566 and 0.545 respectively and QMEAN Z-score was −4.54, −3.36 and −3.66 respectively. Each model was compared to its template to verify that the modeling step had not significantly altered backbone and side chain conformation. [...] For the full-length Kir2.1 channels simulation, the channels were immersed in an explicit palmitoyloleoyl-phosphatidylcholine (POPC) bilayer generated by the VMD membrane package. The whole system was then solvated, and K+ and Cl− of ~150 mM were positioned randomly among the solvent to neutralize the system. We built three systems (Closed, Activated, and Open states). Each system involved ~140,000 atoms in the MD simulation. Full three-dimensional periodic boundary conditions were used. Each system was in a rectangular box with the size of 101 × 98 × 156 Å3 (Closed state), 101 × 99 × 156 Å3 (Activated state), and 104 × 105 × 153 Å3 (Open state), respectively. The solvated systems then underwent four equilibration steps: (i) 4 ns of equilibration with melting of lipid tails, (ii) the entire protein was fixed for 5 ns, enabling reorganization of the lipid and solution (iii) 5-ns extensive equilibration with protein released, (iv) finally letting the system relax freely for over 10–20 ns until reaching equilibrium. Each Targeted Molecular Dynamic is performed on the equilibrated system.ALL MD simulations were performed in with the NAMD2 program (http://www.ks.uiuc.edu/Research/namd/) and the CHARMM 27 force filed. A scaling factor for 1–4 interactions of 1.0 was applied in all simulations. Langevin dynamics and the Langevin piston were used to maintain the temperature at 310 K and a pressure of 1 atm with a damping coefficient of 1 ps–1. The barostat oscillation and damping time scale for the Langevin piston method were set to 200 fs and 50 fs, respectively. The van der Waals interactions were modeled using the Lennard-Jones potential. Short-range interactions was smoothed at 10 Å and truncated at 12 Å. Long-range electrostatic forces were taken into account using the Particle Mesh Ewald (PME) method (120 × 120 × 180 grid points). Non-bonded and PME calculations were performed on every time step. The time step employed was 2 fs, and pair lists were updated every 20 iterations. The coordinates were saved every 4 ps for analysis. Pore dimensions were evaluated using HOLE. For molecular visualization and structural diagrams we used VMD. Simulations were carried out on a 64-processor Linux cluster. […]

Pipeline specifications

Software tools SWISS-MODEL, QMEAN, VMD, NAMD, CHARMM
Applications Small-angle scattering, Protein structure analysis
Diseases Hamartoma Syndrome, Multiple