Extracellular Zn²⁺ occupancy is conformationally coupled to reorganization of electrostatic networks that control voltage-dependent gating. (A and B) Coulombic interactions between ionizable side chains in Hv1 F·Zn²⁺ c0 (A, snapshot taken at t = 110 ns; arrowhead A in Fig. 5 H) and Hv1 F + Zn²⁺ (B, snapshot taken at t = 70 ns; arrowhead B in Fig. 5 F) MD simulations are illustrated by dashed lines. Thick black lines represent strong interactions (distance <3.0 Å), and thin gray lines represent weak interactions (distance >3.0 Å). Helices are represented by tubes (S1) or ribbons (S2–S4) colored as in previous figures. In Hv1 F·Zn²⁺ c0 (A), R1–R3 each participate in strong pairwise interactions with acidic side chains (D112/D^1.51-R1, E153/E^2.53-R2, and D174/D^3.50-R3). In Hv1 F + Zn²⁺, R3 and E^2.53 do not participate in a strong Coulombic interaction (B). (C–E) Calculated distances between selected terminal oxygen (Asp, Glu) and nitrogen (Arg) atoms of side chains that participate in salt bridges identified using the VMD 1.9.2 salt bridge plugin are shown for 120 ns (Hv1 F + Zn²⁺ and Hv1 F·Zn²⁺ c0) or 50 ns (Hv1 F·Zn²⁺ c5) MD trajectories (Fig. 5, E–H). Colored symbols and lines represent specific bonds in each MD trajectory. In C–E, distances are binned at 0.25 Å intervals. (F) Relative occupancies of side chain–to–side chain H-bond pairs (distance cutoff <3.0 Å, angle cutoff 20°, VMD 1.9.3 H-bond plugin) calculated during Hv1 F + Zn²⁺ (stippled blue columns) and Hv1 F·Zn²⁺ before (Hv1 F·Zn²⁺ c5, solid black columns) and after (Hv1 F·Zn²⁺ c0, solid gray columns) relief of harmonic constraints. Asterisks indicate that the H-bond does not form during the indicated MD trajectory. (G) PCC calculated for distance changes between atoms in the indicated salt bridges and ZN¹³ to H^2.40-N_δ1 (black columns) or ZN¹³ to H^3.71-N_δ1 (blue columns) during Hv1 F·Zn²⁺ c5 (hashed columns) and Hv1 F·Zn²⁺ c0 (filled columns) MD trajectories. Asterisks indicate that the indicated salt bridge does not form during the MD trajectory. See Table 3 and Fig. S7 D.