Computational protocol: Modeling the human Nav1.5 sodium channel: structural and mechanistic insights of ion permeation and drug blockade

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Protocol publication

[…] Initially, the full-length amino-acid sequence of the α-subunit of hNav1.5 was dissected into nine protein domains that were independently modeled using the Iterative Threading Assembly Refinement (I-Tasser) software. Transmembrane regions of the channel were built using the crystal structure of NavAB bacterial VGSC (PDB ID: 3RVY). As described in the 2011 Payandeh et al crystal structure of NavAB bacterial VGSC, the deposited X-ray structure (PDB ID: 3RVY, 2.7Å resolution) represents a closed conformation. In 2012, the same group made another breakthrough, where they were able to crystallize the same channel in another conformation that they believed was representative of an inactivated state of the channel (PDB ID: 4EKW, 3.2Å). After superposing the two structures, we realized that there is only a slight shift of the S6 helices terminal, and the overall root-mean square deviation (RMSD) between the two structures was as low as 0.7Å. We decided to henceforth use the 3RVY structure as a template as it has a slightly better resolution.The NavAB ion channel has been extensively used to build mammalian VGSC, such as the hNav1.4 model by Mahdavi and Kuyucak and the hNav1.7 model by Yang et al. The intracellular regions included in the model were either retrieved from the existing crystal structures or were independently built using I-Tasser. Please refer to supplementary information for a detailed description of the homology modeling procedures employed here. Figure S1 represents the sequence alignment between the target hNav1.5 and different template sequences. Cartoon representations of the complete model of hNav1.5 are also given in (side view) and C (top view).The homology model of the hNav1.5 structure was embedded in a palmitoyl-oleyl-phosphatidylcholine (POPC) lipid bilayer and was subjected to ~680 ns-long classical MD simulation. The full system (protein, lipid, water, and ions) was built using the powerful CHARMM-GUI membrane builder module, and using the CHARMM36FF forcefield. The system underwent energy minimization, heating to 310 K over 50,000 steps, with 100 ns-long equilibration, and, finally, production simulation was performed for 680 ns. A weak-restraining force of 0.1 kcal/mol/A2 was applied to the backbone atoms (C, N, and O) of the transmembrane regions during the first 200 ns of production run. All MD simulations were performed using a NAMD2.9 package. […]

Pipeline specifications

Software tools I-TASSER, CHARMM-GUI Membrane Builder, NAMD
Application Membrane protein analysis
Organisms Homo sapiens
Diseases Neural Tube Defects, Genetic Diseases, Inborn
Chemicals Sodium