Computational protocol: Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations

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Protocol publication

[…] A structural model of the KCNQ1 VSD (residues 100 to 249) was generated using the protein structure prediction software package Rosetta (version 3.8) () based on the cryo-EM structure of Xenopus KCNQ1 (PDB ID: 5VMS) (), and sequence alignment was generated with ClustalW (). Four thousand models of KCNQ1 VSD were assembled through comparative modeling () using the Rosetta Membrane energy function (). Gaps in the threaded model resulting from unresolved regions in the template structure were reconstructed by fragment insertion and cyclic coordinate descent loop building (). All models underwent side-chain repacking and all-atom refinement while applying a low constraint to the initial coordinates. Models were clustered on the basis of root mean square deviation (RMSD). The lowest-scoring model of the largest cluster was considered the representative model. Its Cα RMSD compared to the Xenopus KCNQ1 VSD structure was 2.3 Å. MolProbity analysis of the representative model reported an overall score of 1.34 (98th percentile), a clash score of 2.02 (99th percentile), 140 (95.0%) residues in favored regions of the Ramachandran plot, all residues in allowed regions, 126 (98.4%) favored rotamers, no poor rotamers, and no Cβ deviations or bad backbone angles. [...] An MD simulation of the KCNQ1 VSD was performed in an explicit DMPC bilayer at 313 K using Amber16 () and the Lipid14 force field (). Three independent trajectories, each having a total length of 500 ns but starting with different input models, were computed. Our ensemble of KCNQ1 VSD homology models was clustered using a hierarchical full-linkage clustering algorithm (), and the centroids of the three largest clusters were chosen as starting coordinates. The coordinates of each input model were aligned with the membrane normal using the PPM web server (). A complete model of the KCNQ1 VSD in a DMPC bilayer (110 lipids per leaflet) was prepared with the membrane builder tool of the CHARMM-GUI website (). A TIP3P water layer of 20 Å was included, and Cl− ions were added to neutralize the charge of the system. Each bilayer system was first minimized for 15,000 steps using steepest descent followed by 15,000 steps of conjugate gradient minimization. With the KCNQ1 VSD restrained to its starting coordinates, the lipid and water were heated to 50 K over 1000 steps with a step size of 1 fs using constant boundary conditions and Langevin dynamics with a rapid collision frequency of 10,000 ps−1. The system was then heated to 100 K over 50,000 steps with constant volume dynamics and the collision frequency set to 1000 ps−1 and, finally, to 313 K over 100,000 steps with constant pressure dynamics and anisotropic pressure scaling turned on. Positional restraints on the KCNQ1 VSD were then gradually removed over 500 ps, and the system was equilibrated for another 5 ns at 313 K. Production MD was conducted for 500 ns using a step size of 1 fs, constant pressure periodic boundary conditions, anisotropic pressure scaling, and Langevin dynamics. MD trajectories were analyzed using CPPTRAJ (version 15.0) () and VMD (visual molecular dynamics; version 1.9) (). […]

Pipeline specifications

Software tools Clustal W, MolProbity, AMBER, CHARMM, VMD
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens
Diseases Heart Diseases