Model of the extracellular network in putative Hv1 VS domain resting-state model and x-ray structures. (A) Amino acid sequence alignment of the VS domain in C. intestinalis (Ciona), Mus musculus (mouse), C. pelagicus (Coccolith), and Homo sapiens (human) Hv1 proteins. The positions of selected residues in human Hv1 are shown in italics below the sequences, and residue locations in a consensus numbering scheme described previously (Randolph et al., 2016) are shown above the sequences. The positions of R1 (R^4.47), R2 (R^4.50), and R3 (R^4.53) are indicated in blue type. Approximate boundaries of helical segments 1–4 (S1–S4), the C-terminal CC motif, and S4–CC linker helix (5L) are indicated by colored numbers and colored boxes (S1, yellow; S2, green; S3, blue, S4, red; 5L, magenta; CC, gray). Residues are colored type (red, Asp; orange, Glu; aqua, His; blue, Arg or Lys; green, Asn). Asterisks indicate residues that are mutated in this study. (B and C) The positions of candidate extracellular Zn²⁺-coordinating side chains in Hv1 D and Hv1 F are shown in snapshots taken at the beginning (B) or end (C) of respective MD trajectories (Randolph et al., 2016; Fig. 5F). Helical segments are represented as colored ribbons (S1, yellow; S2, green; S3, blue; S4, red; 5L, magenta), loops are shown as silver (Hv1 D) or black (Hv1 F) tubes, and selected residue side chains (labeled) are indicated by colored licorice representations. The distances between C_γ atoms in H140 (H^2.40) and H193 (H^3.71) are 20.32 Å (Hv1 D) and 7.28 Å (Hv1 F). (D) A representation of the mHv1cc x-ray structure (PDB accession no. 3WKV) is shown for comparison to Hv1 D and Hv1 F. (E) A view of Hv1 D (lighter color shades) and Hv1 F (darker color shades) in overlay (structural alignment, MultiSeq STAMP, VMD; Roberts et al., 2006) from within the plane of the membrane illustrates the overall similarity in backbone structure in regions outside the S1–S2 and S3–S4 loops. The S4 and 5L helices are separated by a helical break at G215 (G^4.57).