Nature Structural and Molecular Biology: doi: /nsmb.2783

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1 Supplementary Figure 1: Crystallized chimera construct (mhv1cc). (a) Sequence alignment between mhv1cc and other VSDs. These sequences (mhv1cc, Kv1.2 Kv2.1; shaker family voltage gated potassium channel Kv1.2 Kv2.1 paddle chimera channel, NavAb; voltage gated sodium channel from Acrobacter butzleri, Kv1.2; voltage gated potassium channel subunit Kv1.2, KvAP; voltage gated potassium channel from Aeropyrum pemix, NavRh; NaChBac orthologue from the marine alphaproteobacterium HIB114, and Ci VSP; voltage sensor phosphatase from Ciona intestinalis were aligned using program Clustal W 39. The red characters show the periodically basic residues on S4. The green characters show the hydrophobic residues highly conserved among VSDs. (b) The cartoon diagram of crystallized chimera constructs (mhv1cc). The S2 S3 half intracellular side (Glu149 Phe171) and cytoplasmic coiled coil (Val216 Asn269) of mouse Hv1 or VSOP were replaced with the intracellular portion of Ci VSP (Asp164 Leu188) and the leucine zipper transcriptional activator GCN4 from S. cereviece (Arg249 Arg281), respectively. The intracellular portion of Ci VSP and GCN4 show red and green diagrams, respectively. (c, d) Thermal stability assay of mhv1cc with CPM. (c) A raw fluorescence signal plotted against increasing temperature. (d) Melting temperature (T m ) was determined as the first inflection point by calculating derivative of raw fluorescence shown in a against temperature. Tm of mhv1cc was estimated to be 70.6 ºC. The red and blue show plots with (n = 4 technical replicates) and without mhv1cc (n = 2 technical replicates), respectively. (e, f) Proton current of mhv1cc recorded at the indicated two different ph environments from HEK293T cells. (g) Stoichiometry of mhv1cc in HEK293T cells as shown by cross linking with DSS. The arrow shows a band corresponding to dimer of mhv1cc. (h) H + flux assay using proteoliposome. Data from vesicles with and without mhv1cc were shown in red and black curves, respectively. Error bars depict means ± S.D. (n = 3 technical replicates).

2 Supplementary Figure 2: Zn 2+ binding and S4 position. (a d) Se Met and Zn 2+ anomalous Bijvoet signals. These blue mesh show the initial map phasing by Se MAD at 4.3Å (contoured at 1.0σ level). All Bijvoet anomalous difference signals (green mesh, contoured at 3.0σ level) were calculated by Se Met1(a), Se Met2(b), and Se Met3(c) data collections, respectively. The asterisk shows unknown weak Bijvoet anomalous signal. The red mesh shows the Bijvoet anomalous difference map of Zn 2+ calculated by Cryst Z1 data collection (contoured at 5.0σ level) (d). (e) Electron density map of mhv1cc and anomalous Bijvoet signal of Zn 2+, shown in stereogram (wall-eyed viewing). σ A weighted 2mFo DFc map of mhv1cc (blue mesh) contoured at 1.0σ with the final model shows stick model. The map was calculated using native crystal (Cryst B, S76 mhv1cc) at 3.45Å resolution. The magenta mesh shows the Bijvoet anomalous difference map of Zn 2+ contoured at 5.0σ ( Å). The Zn 2+ anomalous map was calculated from the native crystal data (Cryst Z1) collected at 1.275Å wavelength using the phase as the refined coordinate of mhv1cc. (f) Stereo drawing of long helix (S4 and coiled coil) in mhv1cc. The S4 consists of 3 10 helix (Arg201 Arg204) and α helix (Arg204 Arg207). Three arginine residues (Arg201, Arg204, and Arg207), and Met217 were drawn by stick model. The blue mesh shows the experimental map with MAD phases contoured at 1.0σ. The green mesh shows the Bijvoet anomalous difference map of selenomethionine (Met217) contoured at 5.0σ. The anomalous map of selenomethionine was calculated from MAD phases of Se-Met1 (Table 1). Since cell dimensions of Cryst A were slightly deviated from those of Se- Met1, the refined model was fitted onto the Se MAD map of Se-Met1 by rigid-body refinement. (g i) Representative current traces in the presence of distinct doses of zinc ions. Data sets in each mutant [E115S(g), D119S(h), and ΔNΔC(i)] were recorded from the same patches. ΔNΔC denotes a version of mhv1 in which C-terminus and N-terminus were truncated at position 216 and 77, respectively 16. Currents were elicited by test pulses to 100 mv under ph out /ph in = 7.0/7.0. The holding potential was 60 mv (black curve; 0 μm, red curve; 1 μm, and blue curve; 10 μm). (j, k) Sequence alignment of S4 (j) and comparison of S4 position among VSDs [mhv1cc (this structure, 3WKV)], Kv1.2 [2A79 (ref.2)], KvAP (1ORS 39 ), and NavRh (4DXW 36 ) (k).

3 Supplementary Figure 3: Sequence alignment of Hv1 orthologs from different species. Sequence alignment of Hv1 orthologs from different species. The red characters show the positions of conserved, periodically aligned arginine residues of S4. The orange and blue layers show the hydrophobic residues in a lower hydrophobic layer (HLin) and an upper layer (HLex), respectively. The green layers show the Zn 2+ binding residues. Hv1 sequences of Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr), Xenopus laevis (Xl), Ciona intestinalis (Ci), Coccolithus pelagicu (Cp), Strongylocentrotus purpuratus (Sp, purple sea urchin), are aligned with mhv1cc, using Clustal W 39. In S2 S3 linker of Hv1cc, the length increased by one residue when the original sequence of mhv1 was replaced by the corresponding region of Ci VSP (underlined). Pro184 in Ci VSP corresponds to Pro159 of mhv1cc. In order to avoid confusion, Pro159 (shown by the red asterisk) of mhv1cc was re assigned to Pro158a.

4 Supplementary Figure 4: Comparison of water accessibility profile of mhv1cc with previous findings on mhv1, CiHv1 and hhv1. Comparison of water accessibility profile of mhv1cc with previous findings on mhv1, CiHv1 and hhv1. The symbols + and show the accessible and the inaccessible residues, respectively. The information of accessible or inaccessible sites were cited from several studies of the cysteine scanning with accessibility to MTS or the histidine scanning of zinc sensitivity by electrophysiology. State dependent MTS or Zn 2+ accessibility are shown with three colored characters (blue; site extracellularly accessible to MTS or Zn 2+ in activated state from CiHv1 or hhv1. red; site intracellularly accessible to MTS in resting state from CiHv1. green; site intracellularly accessible to Zn 2+ in activated state from hhv1). # cysteine scanning of accessibility to MTS reagent done by electrophysiology on CiHv1 (Ref.19) histidine scanning of Zn 2+ sensitivity done by electrophysiology on hhv1 (Ref.20) cysteine scanning of accessibility to MTS reagent done by electrophysiology on CiHv1 (Ref.18) accessibility to AMS reagent44,45

5 Supplementary Figure 5: Proposed dimer model of mouse Hv1. (a) The amino acid sequences were represented by the abcdefg convention for the coiled coil. The red characters show residues of dimer interface (a and d). Dimer interface of mhv1cc was predicted from dimer interface of GCN4 leucine zipper and mouse Hv1 cytoplasmic coiled coil (3VMX 28 ). (b) The dimer model of mhv1cc. Superimposition of cytoplasmic coiled coil region of mouse Hv1 (Ile224 Gly266) and mhv1cc (Ile225 Leu242) was performed by least squares superposition of Cα atoms (RMSD = Å). The arrow shows a coiled coil region. The trimer in crystal packing is shown in box.

6 Supplementary Table 1 Additional X-ray data collection statistics. Se Met2 # (L107M L118M) Se Met3 # (L182M) Cryst Z1 Cryst Z2 # Data collection Space group P6 3 P6 3 P6 3 P6 3 P6 3 Cell dimensions a, b, c (Å) 82.9, 82.9, , 83.9, , 85.7, , 82.7, , 82.9, 89.7 α, β, γ ( ) 90.0, 90.0, , 90.0, , 90.0, , 90.0, , 90.0, Wavelength Resolution (Å) ( )* ( )* ( )* ( )* ( )* R merge (0.487) (0.532) (0.827) (0.493) (0.469) I / σi 67.9 (10.9) 40.5 (4.10) 48.4 (2.96) 52.8 (5.22) 23.8(2.75) Completeness (%) 97.9 (96.7) 99.4 (100.0) 99.4 (100.0) 99.6 ( (100.0) Redundancy 26.7 (27.7) 10.7 (9.3) 11.7 (11.7) 11.0 (11.5) 11.1 (11.5) *Values in parentheses are for highest resolution shell. # Data sets were derived from the T57 mhv1cc construct (Cryst-A). Data sets were derived from the S76 mhv1cc construct (Cryst B).

7 Supplementary Note The presence of two hydrophobic layers may have implications in proton permeation. Minimum S4 motion upon activation could allow protons to be conducted by recruiting water molecules in the cavity to the entire water wire across the channel whereas proton leakage is prevented in resting state. In other words, double hydrophobic layers may ensure that proton leakage does not occur in resting state, whereas even small change of the position of S4 will allow proton flow. This may resemble the case of M2 proton channel of influenza virus which also shows two barriers, built by His37 and Trp41 in tetramer structure in closed state 1. The cavity in M2 proton channel formed between His box and Trp basket constrictive cluster accommodates water molecules in M2 proton channel. Besides this significance in proton conduction, double hydrophobic layers may also have implications in mechanisms of voltage dependent gating. Upon motion of S4 associated with membrane depolarization, cancelling or modification of one of two hydrophobic layers may alter focused electric field around voltage sensing charged residues on S4. This may enable significant transfer of gating charge with smallest structural change by a mechanism similar to that previously proposed as transporter model of voltage sensing in Kv channels 2, 3. References for supplementary note 1. Schnell, J.R. & Chou, J.J. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, (2008). 2. Ahern, C.A. & Horn, R. Focused electric field across the voltage sensor of potassium channels. Neuron 48, (2005). 3. Posson, D.J., Ge, P., Miller, C., Bezanilla, F. & Selvin, P.R. Small vertical movement of a K + channel voltage sensor measured with luminescence energy transfer. Nature 436, (2005).