Finally, these averaged coordinates were used as target coordinates for Khalili-Araghi’s initial resting-state model, and a TMD simulation was performed. This final step was done to ensure that the nonphysical averaging of the
coordinates did not produce an energetically unfavorable conformation. The relative rmsd among the four models do not exceed 2.2 Å, suggesting that the magnitude of the charge movement within the VSD is not expected to differ markedly from the previous MD calculations by Khalili-Araghi et al. (2010). An animation rotating the superimposed models along the z axis is given in Movie S1. To accurately compare the active and resting states, we aligned the tetramer structures provided by Khalili-Araghi with Protease Inhibitor Library their principal axis along the membrane normal axis. To lend weight to each of the subunits, we imposed 4-fold symmetry on the full-length tetrameric channels by performing a spatial average on the carbon alpha atoms. The Oriented Proteins in Membranes database was consulted for placing the tetramers vertically in the membrane by making sure the S1 and S2 helices exhibited no vertical movement (Lomize et al., 2006). Selumetinib molecular weight Finally, the isolated VSD was aligned to the VSD of the resting-state conformation of the symmetric tetramer for the comparison shown in Figure 3. It is noted that the isolated
VSD in the membrane is tilted with respect to the orientation observed in the full-length tetrameric channel. The biotin-avidin trapping model system was generated using a multistage protocol. The complex was assembled using restrained MD and then relaxed for 2 ns. First, residue 298 was mutated to cysteine to conjugate the biotinylated linker, as was done in
Ruta et al. (2005). Next, the avidin (1 AVD) was oriented along z and kept rigid while being steered toward the VSD in vacuum. A restraint was used at z = −12 Å to prevent the avidin from penetrating the membrane region. The rigid body constraints on the avidin were removed when the structure was in close proximity to the VSD. The antechamber package was used to generate force-field parameters required for simulating the biotinylated linker (Wang et al., 2006). Harmonic restraints Org 27569 were then used to slowly steer the linker toward residue 298 and to keep the biotin end bound to avidin. Finally, CHARMM-gui provided scripts to generate the all-atom explicit water/lipid membrane system (Jo et al., 2008). The resulting model is shown in Figure 4. All figures were generated by the molecular visualization package VMD (Humphrey et al., 1996). The authors would like to thank Luca Maragliano for valuable discussions, Fatemeh Khalili-Araghi for providing the refined initial models, and Amelia Randich for helpful manuscript revisions. This work was supported by the National Institutes of Health via grant GM062342 (B.R.), grant GM030376 (F.B.), and training grant GM007183-35 (E.V.).