Fig. 4: Molecular dynamics simulations reveal water wires, Cl⁻ leaving, and inner-gate opening.

a Simulation snapshot depicting water wires that connect to the intracellular solution. b Cartoon model of proton transfer from E_gate to the intracellular solution along water wire 2. The resulting negatively charged E_gate is predicted to return to the anion pathway, expelling two Cl⁻ ions. c Cl⁻ leaves during eight out of ten 1.5-µs simulations with E_gate deprotonated. Each plot shows the distance from inner-gate residue Y445 to the nearest Cl⁻ ion; >12 Å indicates that Cl⁻ no longer occupies anion-binding sites with the pathway. Unsmoothed traces (light purple lines) and traces smoothed with a moving average (dark purple lines) are shown for all simulations. The time traces were smoothed using a moving average with a window size of 20 ns. d Simulation snapshots showing a representative Cl⁻-leaving event. Concomitant with Cl⁻ leaving to the extracellular side (i.e., away from the viewer), inner-gate residues S107 and Y445 move away from one another, opening the inner gate. Cartoon depictions are shown beneath each snapshot. e Water wires occurred with similar abundance in E_gate protonated and deprotonated simulations, indicating that Cl⁻ binding does not substantially influence water-wire formation. Each dot corresponds to one simulation and shows the fraction of frames in which any of the three classes of water wires is formed. The middle line of each box in the plot is the median across simulations, with the box extending from the 1st to the 3rd quartile and defining the interquartile range. Whiskers extend to the last data points that are within 150% of the interquartile range. Note that Cl⁻ is always bound in simulations with E_gate protonated, whereas no Cl⁻ is bound in over 70% of simulation frames with E_gate deprotonated. A simulation snapshot showing water wires in the absence of Cl⁻ is shown in Supplementary Fig. 10.