MD simulations of Zn²⁺-bound (Hv1 F·Zn²⁺) and unbound (Hv1 F + Zn²⁺) Hv1 F model structure. (A–C) Snapshots taken from Hv1 F·Zn²⁺ (A: t = 110 ns; see G and H) and Hv1 F + Zn²⁺ (B: t = 70 ns; see F) MD simulations are shown alone or in overlay (C). Zn²⁺ ions are represented as colored spheres (A: ZN¹³, violet; B: ZN² and ZN¹³, cyan). In A, ZN¹³ is coordinated by H140/H^2.40-N_δ1 and H193/H^3.71-N_δ1, E119/E^1.58-O_ε1 and E119/E^1.58-O_ε2, and two water molecules (W¹⁰⁸⁰ and W²¹³, red/white CPK representations). In B, the first Zn²⁺ solvation shells of ZN² and ZN¹³ are each stably coordinated by six water oxygen atoms that are on average 2.100 ± 0.004 Å and 2.100 ± 0.002 Å (mean ± SD) away from the respective Zn²⁺ ion (Fig. S3); ZN² and ZN¹³ are separated by a mean distance of 6.88 ± 0.68 Å (mean ± SD measured between t = 50–70 ns of Hv1 F + Zn²⁺). In A–C, helices, loop segments, and selected side chains are represented as in Fig. 2; S1–S2 and S3–S4 loop backbones are colored ochre (Hv1 F·Zn²⁺) or magenta (Hv1 F + Zn²⁺). Except for representations of H^2.40 and H^3.71 imidazole groups, hydrogen atoms are omitted for clarity. Water molecules outside the first solvation shells and lipids are not depicted. (D) Distance histograms for ZN¹³ to H^2.40-N_δ1 (open circles) or H^3.71-N_δ1 (filled circles) are calculated for 120 ns (Hv1 F + Zn²⁺, cyan circles; Hv1 F·Zn²⁺ c0, magenta circles) or 50 ns (Hv1 F·Zn²⁺ c5, purple circles) MD trajectories (F and H). First solvation shell interactions (peak at ∼2.1 Å) are not observed in Hv1 F + Zn²⁺, but second shell (∼4.6 Å peak) and third shell (∼6.5 Å peak) are observed in all systems. (E and F) Distances between selected terminal carbon (Arg, Asp, Glu, His, and Phe) or nitrogen (Arg and Lys) atoms during a 100-ns MD simulation of theHv1 F + Zn²⁺ system are plotted in function of simulation time. Atomic distances over the entire (F) or initial 5 ns (G) of the MD trajectory are represented by colored lines. (G) Distances between terminal carbon or nitrogen atoms of selected side chains are plotted in function of Hv1 F·Zn²⁺ MD simulation time. Atomic distances over the entire simulation are represented by colored lines. The Hv1 F·Zn²⁺ system was initially equilibrated with 5 kcal/mol/Å harmonic constraints applied to bonds between ZN¹³ and both H^2.40-N_δ1 and H^3.71-N_δ1 (t = 0–50 ns: c5, blue bar). The harmonic constraints were reduced to 3 kcal/mol/Å for 20 ns (t = 50–70 ns: c3, green bar) and removed for the remaining 110 ns of simulation time (t = 70–180 ns: c0, white bar). (H) Distances between ZN¹³ and selected atoms in its first solvation shell are plotted in function of Hv1 F·Zn²⁺ MD simulation time after relief of harmonic constraints between ZN¹³ and both H^2.40-N_δ1 and H^3.71-N_δ1 (Hv1 F·Zn²⁺_c0, see H). Inset: Atomic distances between ZN¹³ and the indicated atoms with expanded scaling.