Sodium ion binding and salt-bridge stability in MD simulations with different protonation states of Asp163, Asp164, and Lys300. (Left column; A and B) Asp163 deprotonated and Asp164 and Lys300 protonated (simulation S1/1, protomer B). (Middle column; C and D) Asp163 and Asp164 deprotonated and Lys300 protonated (simulation S2/1, protomer B). (Right column; E and F) Asp163, Asp164, and Lys300 deprotonated (simulation S4/1, protomer B). (Top row; A, C, and E) Distances between the closest sodium ion and Asp163 or Asp164 are plotted as a function of time. Spontaneous Na⁺ binding to Asp164 was observed when both aspartates were deprotonated. (C) Continuation of the simulation with Lys300 deprotonated (a 3-ns equilibration simulation with position restraints on all heavy protein atoms is symbolized by dashed lines between panels) leads to a rapid change in the Na⁺-binding mode toward closer interaction with Asp163. (Bottom row; B, D, and F) Distance of the closest Asp163 carboxyl group from the N-amino group of Lys300. Distances <4 Å are indicative of a stable salt-bridge interaction (yellow shaded area), whereas those ≥4 Å are considered a weak or broken salt bridge. Binding of Na⁺ to Asp164 destabilizes the salt bridge. (D) Lines show data averaged over blocks of 10 ns, with fluctuations in the data indicated as shaded regions encompassing the lower 5 and upper 95% percentile. The snapshots show the Na⁺ binding event with subsequent rupture of the salt bridge (cytoplasmic view along the axis of helix TM 5). Videos 1 and 2 show this simulation. Repeat simulations (see Table 1) are shown in Figs. S3–S7.