Fig. 6: Molecular dynamics analysis of hydration and Na+ permeation through the rNaV1.5C/LqhIII complex.
a Side view of rNaV1.5C (orange ribbons; domains II and IV) from MD simulations highlighting Na+ ions (blue spheres), the water-occupied volume within a cylinder of radius 8.5 Å (red surface), and the protein-occupied volume within a cylinder of radius 12 Å (colorless surface). In this snapshot, the protein cavity (outlined in a black rectangle) at the intracellular activation gate (ICAG; purple shaded region, −2.8 nm < z < −1.5 nm) is dehydrated. The QuickSurf representation in VMD was used for surfaces. b Average hydration along the pore-axis for simulations of rNaV1.5C/LqhIII (red line) with and (black line) without the toxin. Overall pore hydration was unchanged whether or not the toxin is included in simulations. Shading corresponds to the standard error of the mean (s.e.m.). c Molecular representations of the gate containing Nwater = 3 (left) or 15 (right) water molecules. d Average probability distribution of Nwater in the gate. The gate is more likely to contain 10 or more water molecules when the toxin is present. Data points and shading represent mean and s.e.m. e Bottom (intracellular) view of the activation gate for cryo-EM rNaV1.5C (PDB ID: 6UZ3; cyan), cryo-EM rNaV1.5C/LqhIII (yellow), and an example conformation from the most sampled basin in MD (magenta) are superimposed. The distances d1, d2 between opposing helices are shown schematically (see “Methods”). f Free energy of d1 vs. d2 computed from MD simulations. The reference cryo-EM structures of (cyan+) rNaV1.5C and (yellow×) rNaV1.5C/LqhIII are indicated. Contour lines are shown every 0.5 kcal/mol from 0 to 4 kcal/mol. In the simulations, the intracellular activation gate often contracts and adopts an asymmetric conformational basin with d2 < d1, with the symmetric conformation observed by cryo-EM corresponding to a metastable state 0.5 to 1 kcal/mol higher in free energy. Time frames spread across 30 independent simulations of rNaV1.5C with or without LqhIII were used for analyses (n = 29,026 frames for rNaV1.5C and n = 29,899 frames for rNaV1.5C/LqhIII).
