Figure 6. Comparison of resting- and activated-state Hv1 VS domain model structures.

(A) Hv1 B was mutated (N4R) in silico and subjected to energy minimization to demonstrate the possible position of the N4R side chain in a VS-activated (G_AQ-blocked) conformation. Other atomic positions are not appreciably different from Hv1 B. S2 (green) and S3 (blue) helices are represented by colored tubes; S4 is shown as a red ribbon and S1 is not shown. Side chains of D112 (D^1.51), D185 (D^3.61), R3 (R^4.53) and N4R (N^4.56R) are shown in the colored licorice ‘element’ scheme (carbon, cyan; oxygen, red; nitrogen, blue) and the F150 (F^2.50) side chain is white. Distances (in Å) between selected carboxylate oxygen atoms in D112 or D185 and either R3 nitrogen atoms or the R3 C_α atom are indicated by dashed arrows. (B) Positions of selected residue side chains in the Hv1 E mutant model structure (produced by in silico R1H mutation of Hv1 D) are superimposed on Hv1 B N4R shown in A. D112, D185 and R1H side chains are represented by ‘brushed metal’ coloring of licorice element representations; F150 is gray. The S4 helix in Hv1 E is shown as a gold ribbon; other helices are as shown in A. The dashed arrow indicates the distance (in Å) between the C_α atoms of D185 and R1H in Hv1 E. (C–F) Backbones of Hv1 D (C), Hv1 E (D), Hv1 B (E) and the Hv1 B N4R mutant (F) model structures are represented by thin (S1-S3) or thick (S4) colored ribbons and inter-helical loop regions are represented by gray tubes. Selected residue side chains are shown in colored licorice (D112/D^1.51, red; F150/F^2.50, gray or white; D185/D^3.61, magenta; R1/R^4.47, cyan; R2/R^4.50, blue; R3/R^4.53, violet; N4/N214/N^4.56, green; N4R, cyan/blue). Structures are vertically aligned by the position of the F150/F^2.50 C_α atom. Labels indicate the predicted functional state of the protein that correspond to the depicted structure. In C–F, helices are colored yellow (S1), green (S2) and blue (S3) and inter-helical loop regions are not shown for clarity; S4 residues 202–214 are colored red (Hv1 B), copper (Hv1 D) or gold (Hv1 E). Video 4 shows Hv1 B activated- and Hv1 D resting-state model structures in rotation.

DOI: http://dx.doi.org/10.7554/eLife.18017.022

Figure 6—figure supplement 1. Atomic distances in resting- and activated-state Hv1 VS domain model structures.

(A–C) Hv1 B (A) and Hv1 B N4R (B) model structures are shown individually and superimposed (C). Helices are represented as colored ribbons (S1, yellow; S2, green; S3, blue; S4, red) and selected side chains are shown in colored licorice (D112/D^1.51, red; E119/E^1.58, orange; F150/F^2.50, gray; D185/D^3.61, magenta; R1/R^4.47, cyan; R2/R^4.50, blue; R3/R^4.53, violet; N4/N^4.56, green; N4R, cyan/blue). (D) Atomic distances between selected atoms in Hv1 D N4R that are predicted to participate in salt bridges (cutoff distance = 5 Å, VMD 1.9.2) are plotted in function of time during the final 10 ns of an all-atom MD simulation. (E) Distances between C_α (CA) atoms of selected residues (D112/D^1.51, F150/F^2.50, D185/D^3.61 and N4R) in Hv1 B N4R are plotted in function of time during the last 10 ns of an all-atom MD simulation.

DOI: http://dx.doi.org/10.7554/eLife.18017.023

Figure 6—figure supplement 2. Comparison of resting- and activated-state Hv1 model structures.

(A, B) Ribbon diagrams show Hv1 B activated-state (A) and Hv1 D resting-state (B) model backbone structures in side view. (C) Hv1 B and Hv1 D backbone structures are superimposed. S1-S3 helices are represented by colored ribbons and S4 segments are shown as solid cylinders. (D, E) Side view overlays of Hv1 D (WT) and Hv1 E (R1H) resting-state model structures (D) or Hv1 B (WT) activated- and Hv1 E (R1H) resting-state model structures (E). Video 4 shows Hv1 B activated- and Hv1 D resting-state model structures in rotation.

DOI: http://dx.doi.org/10.7554/eLife.18017.024

Figure 6—figure supplement 3. Comparisons of putative resting- and activated-state Hv1 and Ci VSD model and X-ray structures.

(A, B) Ribbon diagrams of selected VS domain model structures are viewed side-on (A) or from the extracellular space (B). S4 helices are shown as colored solid cylinders (A) or thick ribbons (B). S1-S3 helices are similarly colored in all structures (S1, yellow; S2, green; S3, blue) and S4 helices are colored differently for each structure: Hv1 B, yellow; Hv1 D, blue; Hv1 E, red; Hv1 R2D, lime green; Hv1 r2vz, orange; Hv1 FL, violet; Ci VSD_U, pink; Ci VSD_D, cyan. The side chains of Hv1 D112 (D^1.51, red), Hv1 D185 (D^3.61, magenta) and R1 (R^4.47, cyan) in Hv1 D only are shown in colored licorice; Hv1 F150/Ci VSP F161 (F^2.50) is colored white in activated-state structures and gray in resting-state structures. The structure of the protein backbone in inter-helical loop regions of Hv1 D only is shown as a gray line in A but omitted from B for clarity. (C) Insets show extracellular views of Hv1 B (top left), Hv1 D (top right), Hv1 FL (bottom left) and Hv1 E (bottom right). Helices and side chains are colored as in B. (D) Representations of the S4 helix (Hv1 residues 205–214; Ci VSP residues 223–232) from the indicated structures are shown as thick colored ribbons. The orientations of S4 segments relative to one another are shown as in A. S4 segments are colored as indicated by the labels: Hv1 B, red; Hv1 D, orange; Hv1 E (R1H), gold; Hv1 R2D, lime green; Hv1 r2vz, ice blue; Hv1 FL, purple; Ci VSD_U, gray; Ci VSD_D, cyan; 3RVY, white; 2R9R, light purple; 4DXW, green. Side chains of selected residues are shown in colored licorice: R1 (R^4.47), cyan; R2 (R^4.50), blue; R3 (R^4.53), violet; R4 (R^4.56) and N4R, aqua; N4 (N^4.56), green; Hv1 D185 (D^3.61), magenta; Ci VSP T201 (T^3.65), green; Hv1 D112/ Ci VSP D129 (D^1.51), red; Hv1 F150/ Ci VSP F161 (F^2.50) side chains are white in activated- and gray in resting-state structures. Distance (in Å) measured between R1 C_α atoms in the structures shown is indicated by dashed black arrows. Dashed gray lines indicate vertical position of the indicated resting-state Hv1 F150 C_α atoms.

DOI: http://dx.doi.org/10.7554/eLife.18017.025

Figure 6—figure supplement 4. Atomic distances between backbone C_α atoms in resting and activated Hv1 model structures.

(A) Columns represent distance (in Å) measured between R1 (Hv1 R205, Ci VSP R223; cyan), R2 (Hv1 R208, VSP R226; blue), R3 (Hv1 R211, Ci VSP R229; violet), N4/R4 (Hv1 N214, Ci VSP R232; aqua), D112/129 (Hv1 D112, Ci VSP D129; red) or F150/161 (Hv1 F150, Ci VSP F161; gray) C_α atoms in Hv1 D compared to the indicated structure. Structures were collectively aligned using MultiSeq prior to RMSD measurement. (B) Columns represent differences in the 3-D (x, y, z) coordinates of R1 C_α atoms (in Å) measured between the indicated resting- vs. activated-state VS domain X-ray and model structures. The x, y and z positions of the R1 C_α atoms in Hv1 B, Hv1 D, Ci VSD_U and Ci VSD_D were determined after a structural alignment (MultiSeq) of the indicated pair of structures. Note that differences in the position of R1 in either Hv1 D or Ci VSD_D (pdb: 4G80) resting-state structures to the Ci VSD_U (pdb: 4G7V) activated-state occurs mainly in the z (i.e., vertical or normal to plane of the membrane) axis, but displacements in all 3 axes are observed when comparing the Hv1 B activated-state structure to either Hv1 D or Ci VSD_D resting-state structures. (C) Columns represent distance (in Å) measured between the backbone positions of S1 (yellow), S2 (green), S3 (blue) or S4 (red) helical segments in Hv1 D and the indicated model or X-ray structure. RMSD of C_α atoms in amino acid positions (S1, yellow: Hv1, 104–119; Ci VSP, 121–136; S2, green: Hv1, 140–157; Ci VSP, 151–168; S3, blue: Hv1: 172–185; Ci VSP: 188–201; S4, red: Hv1, 205–214; Ci VSP: 223–232) are compared for Hv1 D and the indicated structures. (D) Columns represent distances (in Å) between D185 (Hv1 D^3.61) or T201 (Ci VSP T^3.65) and R1 C_α atoms in the indicated structure. Note that with the exception of Hv1 FL and mHv1cc, the R1-D185 distance is shorter in activated-state structures. (E–H) Hv1 D (E), mHv1cc (F), Ci Hv1 (G) and Hv1 FL (H) backbone structures are represented by colored tubes (S1, yellow; S2, green; S3, blue) or ribbons (S4: Hv1D, orange; mHv1cc, black; Ci Hv1 ice blue; Hv1 FL, purple). Selected residue side chains are shown in colored licorice using human Hv1 numbering, as indicated by labels: D112/D^1.51, red; F150/F^2.50, gray; D185/D^3.61, magenta; R1/R^4.47, cyan; R2/R^4.50, blue; R3/R^4.53, violet; L204/L^4.46, yellow. Atomic distances (in Å) between D185 C_α (CD) and R1 C_α (CE) atoms are indicated by solid lines. Video 4 shows superimposed Hv1 D and Hv1 FL model structures in rotation.

DOI: http://dx.doi.org/10.7554/eLife.18017.026