SUPPLEMENTARY INFORMATION

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1 doi: /nture10735 KCNH ERG herg3 merg2 herg1 SpIH rhcn3 hhcn1 mhcn2 rhcn4 HCN zelk ELK helk1 helk2 melk3 hcngb3 CNGB1 meag1 meag2 EAG CNGA1 CNGA3 CNGA2 hcnga4 CNG Reltive Conductnce (G/Gmx) Voltge (mv) c zelk helk1 helk2 herg1 meag1 CNGA1 mhcn2 C-linker CNBD A B C D E F A 1 2 zelk helk1 helk2 herg1 meag1 CNGA1 mhcn B C 9 Supplementry Figure 1. Similrity of zelk chnnels to relted ion chnnels., Phylogenetic tree, computed with Colt, showing the reltionships of KCNH, CNG nd HCN ion chnnel fmilies., Conductnce-voltge reltions for zelk chnnels in the sence (lck) nd presence of 1 mm camp (red). Error rs indicte SEM (n=7). c, Amino cid sequence lignment of the C-terminl region of zelk nd other ion chnnels in the KCNH, CNG nd HCN ion chnnel fmilies. The C-linker/CNBHD of zelk chnnels shres 83% mino cid sequence identity with helk1 nd 67% sequence identity with helk2 chnnels. It lso shres 42% identity with herg nd 36% identity with meag chnnels, ut < 26% identity with HCN nd CNG chnnels. Brs represent α-helices nd rrows β-strnds from the zelk structure. 1

, Stereo view of the lignment of the rion representtions of the CNBHD of zelk (the αc helix is green nd the rest of the CNBHD is lue) nd HCN2 (grey) chnnels10.

2 αa αa β9 αb β9 αb αc αc L674 L674 N697 Y740 L682 I683 N697 Y740 L682 I683 L742 G684 L742 G684 Supplementry Figure 2. Stereo view of the structurlly ligned CNBHD of zelk nd HCN2 chnnels, nd residues in the β-roll cvity directly intercting with the intrinsic lignd., Stereo view of the lignment of the rion representtions of the CNBHD of zelk (the αc helix is green nd the rest of the CNBHD is lue) nd HCN2 (grey) chnnels10. camp in the HCN2 structure is yellow., Stereo view of the residues in the β-roll cvity directly intercting with residues Y740 nd L742 of the intrinsic lignd. camp in the structurlly ligned, ut not shown, CNBD of HCN2 chnnels is shown in yellow. Dshed lines show oth polr nd non-polr interctions. 2

zelk / MlotiK unlignded.. 626-738 / 241-349 RMSD: 6.4 Å zelk / MlotiK unlignded (NMR).. 626-738/ 241-355 RMSD 6.6 Å c zelk / MlotiK + camp.

-c, Alignment of the rion representtions of the CNBHD of zelk nd unlignded crystl structure of the CNBD of MlotiK1 chnnels with R348A muttion²⁸ (),

The αc helix of the CNBHD of zelk is green nd the rest of the CNBHD is lue. The CNBD of MlotiK1 is pink. camp in the MlotiK1 structure is yellow.

3 zelk / MlotiK unlignded / RMSD: 6.4 Å zelk / MlotiK unlignded (NMR) / RMSD 6.6 Å c zelk / MlotiK + camp / RMSD 7.3 Å Supplementry Figure 3. Structurl comprison of the CNBHD of zelk nd CNBDs of MlotiK1 chnnels. -c, Alignment of the rion representtions of the CNBHD of zelk nd unlignded crystl structure of the CNBD of MlotiK1 chnnels with R348A muttion²⁸ (), unlignded NMR structure of the CNBD of MlotiK1²⁹ () nd camp-ound crystl structure of the CNBD of MlotiK1 chnnels²⁸ (c). The αc helix of the CNBHD of zelk is green nd the rest of the CNBHD is lue. The CNBD of MlotiK1 is pink. camp in the MlotiK1 structure is yellow. The corresponding PDB ccession numers for the MlotiK1 structures re 1U12 for (), 2XKL for () nd 1VP6 for (c). The RMSDs for the α crons nd the stretch of residues used for the RMSD clcultions re indicted. 3

T696 R591 C584 T696 R591 C584 V667 N697 T592 G581 S685 S687 V667 N697 T592 G581 S685 S687 V564 G684 V564 G684 A675 M572 E582 A675 M572 E582 L574 L574 L677 L677 Supplementry Figure 4.

4 T696 R591 C584 T696 R591 C584 V667 N697 T592 G581 S685 S687 V667 N697 T592 G581 S685 S687 V564 G684 V564 G684 A675 M572 E582 A675 M572 E582 L574 L574 L677 L677 Supplementry Figure 4. Structurl lignment of the β-roll cvity of the CNBHD of zelk (lue) nd of the CNBD of HCN2 (grey) chnnels. Residues directly intercting with camp in the CNBD of HCN2 chnnels nd their corresponding residues in the CNBHD of zelk chnnels re shown in sticks. Residues R632 nd I636 in HCN2 chnnels were omitted from the figure ecuse there re no corresponding residues in the sme reltive loction in zelk chnnels. 4

doi:10.1038/nture10735 Y740 Y740 L742 N741 L742 N741 Supplementry Figure 5. Intrinsic lignd electron density.

5 doi: /nture10735 Y740 Y740 L742 N741 L742 N741 Supplementry Figure 5. Intrinsic lignd electron density. A 2Fo-Fc omit electron-density mp of residues Y740-L742 of the intrinsic lignd ound inside the β-roll cvity of the CNBHD of zelk chnnels. The mp is contoured t 1.0 σ. 5

6 zelk 2 na 50 ms zelk-y740a 2 na 50 ms zelkδ pa 50 ms V 1/2 (mv) zelk Y740A Δ Supplementry Figure 6. Muttions of the β9 strnd shift the voltge dependence of ctivtion to more depolrized voltges., Currents from wild-type, Y740A nd Δ mutnt zelk chnnels. Currents were elicited y pplying series of 100 ms voltge pulses (rnging from -140 to +140 mv in 20 mv increments), followed y 150 ms til pulse to -100 mv., Box plot of V 1/2 vlues for wild-type, Y740A mutnt, nd Δ mutnt zelk chnnels. The line in the middle of the ox represents the medin, the ox represents the 25 th nd 75 th percentiles, nd the whiskers represent the 5 th nd 95 th percentiles of the dt. For oth muttions, the V 1/2 vlues for ctivtion were significntly lrger (P < 0.01, Student s t-test) thn the V 1/2 of wild-type chnnels (wild type: mv, n = 18; Y740A: mv, n = 19; Δ : mv, n = 11). 6

Supplementry Figure 7. Stereo view of the elow-on-the-shoulder interfce of zelk chnnels. The elow is lue nd the shoulder of the neighoring suunit is yellow.

7 Supplementry Figure 7. Stereo view of the elow-on-the-shoulder interfce of zelk chnnels. The elow is lue nd the shoulder of the neighoring suunit is yellow. The residues forming the network of interctions etween the elow nd the shoulder regions re shown in stick. Intersuunit contcts of less thn 4 Å distnce re shown s dshed lines for oth polr nd non-polr interctions. 7

ssymmetric unit nd for oth spce groups of zelk crystls.

8 90 o Supplementry Figure 8. The structures nd dimeric ssemly re preserved for different monomers in the ssymmetric unit nd for oth spce groups of zelk crystls., Alignment of the ckone α-cron trces of the C-linker/CNBHD monomers A (lue) nd B (red) in C2221 spce group., Alignment of dimers formed y the C-linker/CNBHD of zelk chnnels in C2221 (lue) nd P1211 (green) spce groups viewed perpendiculr (left) nd prllel (right) to the two-fold xis. 8

9 monomer dimer GFP Supplementry Figure 9. GFP-tgged C-linker/CNBHD of zelk chnnels dimerizes t sufficiently high concentrtions in solution., FSEC profiles of the GFP-tgged C-linker/CNBHD of zelk chnnels for the indicted reltive concentrtions of the smple. The elution volumes of the dimer nd monomer of the C-linker/CNBHD, s well s of the cleved GFP, re indicted y the rrows., Plot of the frction of the dimeric complex versus the reltive concentrtion of the smple. 9

Supplementry Figure 10. Crtoons illustrting possile dimer-of-dimers model nd four-fold tetrmer model for the rrngement of the C-linker/CNBHDs in the full-length zelk chnnel.

2 (PDB 2A79)43 chnnel docked to dimer-of-dimers model () nd four-fold tetrmer model () of the C-linker/CNBHDs of zelk chnnels viewed perpendiculr (top) nd prllel (ottom) to the Kv 1.

These monomers were then rrnged with four-fold symmetry round the Kv1.2 pore xis, s seen in HCN2 (PDB 1Q5O).

10 doi: /nture10735 dimer-of-dimers model four-fold tetrmer model 36 Å 33 Å 90 o 90 o 19 Å c 36 Å 23 Å Å 33 Å Å MthK zelk KcsA 19 Å d MthK zelk KcsA 23 Å 12 Å 30 Å 26 Å 12 Å 23 Å 26 Å Supplementry Figure 10. Crtoons illustrting possile dimer-of-dimers model nd four-fold tetrmer model for the rrngement of the C-linker/CNBHDs in the full-length zelk chnnel. -, A crtoon representtion of two digonlly opposed inner-helices of the Kv1.2 (PDB 2A79)43 chnnel docked to dimer-of-dimers model () nd four-fold tetrmer model () of the C-linker/CNBHDs of zelk chnnels viewed perpendiculr (top) nd prllel (ottom) to the Kv 1.2 pore xis. The Kv1.2 structure ws omitted in the lter for clrity. The dimer-of-dimers model ws uilt y rrnging two zelk dimers with two-fold symmetry round the Kv1.2 pore xis. The four-fold tetrmer model ws uilt y modeling the C-linker from zelk fter the C-linker from HCN2 (PDB 1Q5O)10 using SWISS-MODEL44, nd ttching the CNBHD of zelk. These monomers were then rrnged with four-fold symmetry round the Kv1.2 pore xis, s seen in HCN2 (PDB 1Q5O). The pink-colored spheres represent the mino-terminl residues of ech of the zelk monomers in the models. The modeling ws done in Pymol37. (c,d) A schemtic digrm compring the proposed rrngements of the mino-termini of the C-linker/CNBHDs of zelk for dimer-of-dimers model (c) nd four-fold tetrmer model (d) with the corresponding rrngement for the croxy-terminl residues of the inner helices of KcsA (PDB 1K4C)41 nd MthK (PDB 1LNQ)42 chnnels. 10

11 Supplementry Tle 1: Dt collection nd refinement sttistics Crystl ID T141 (SeMet) T42 (Ntive) T26 (Ntive) T84 (Ntive) Accession codes 3UKN 3UKT 3UKV Dt collection Spce group C222 1 C222 1 P P Molecules / ACU Cell dimensions,, c (Å) 58, 95.3, , 95.5, , 107.8, , , α, β, γ ( ) 90, 90, 90 90, 90, 90 90, 97.38, 90 90, 97.51, 90 Wvelength (Å) Resolution (Å) ( ) ( ) ( ) ( ) R merge (%) 8.1 (48.6) 6.2 (43.4) 4.1 (20.0) 6.6 (45.3) I/σI 13 (2.5) 9.9 (2.4) 12.5 (2.5) 8.1 (1.9) Completeness (%) 97.4 (98) 97.6 (85.4) 93.1 (66.3) 99.8 (99.7) Redundncy 3.7 (3.7) 4.4 (2.0) 3.2 (2.1) 3.5 (3.1) Refinement Resolution (Å) No. reflections R work/ R free (%) / / / No. toms Protein Wter B-fctors Protein Wter R.m.s devitions Bond lengths (Å) Bond ngles (º) Highest resolution shell is shown in prenthesis. 5% of dt ws set side for clcultion of R free. 11

12 SUPPLEMENTARY DISCUSSION The C-linker/CNBHD of zelk chnnels forms dimers insted of tetrmers s oserved in HCN chnnels 10. A dimeric ssemly of the CNBDs ws lso oserved in the crystl structure of the CNBDs of cteril MlotiK1 chnnels, however, the C-linker of the MlotiK1 chnnels consists of only 5 mino cids nd is missing the entire elow-on-the-shoulder region chrcteristic of the eukryotic chnnels 28. Interestingly, the C-linker/CNBD of HCN2 chnnels fvors dimer over tetrmer formtion in solution in the sence of camp 10, lthough the multimeriztion is reltively low ffinity. It hs een suggested tht in the unlignded stte HCN chnnels function s dimer-of-dimers 40. Wht is the quternry stte of the C-linker/CNBHD in intct zelk chnnels? In principle, the C-linker/CNBHD could form either dimer-of-dimers (Supplementry Fig. 10) or tetrmer in intct chnnels (Supplementry Fig. 10). In sequence lignments, the minoterminl end of the C-linker in the structure of zelk is within out six mino cids from the croxy-termini of the pore-lining inner helices in the structures of KcsA (PDB 1K4C) 41, MthK (PDB 1LNQ) 42, nd Kv1.2 (PDB 2A79) 43 chnnels. The spcing of the mino termini in the dimer of zelk (~ 19 Å) closely mtches the spcing of the croxy-termini of the djcent inner helices of Kv1.2 (~ 19 Å) nd is intermedite etween KcsA (~ 12 Å) nd MthK (~ 26 Å) chnnels. However, in order to ccommodte two dimers with pproprite spcing, the dimers would hve to e positioned with their mino termini fcing ech other t distnce of ~ 30 Å etween the mino-termini of the djcent suunits on different dimers (Supplementry Fig. 10 nd c). Alterntively, when ttched to the S6, the C-linker might ssume conformtion closer to the one seen in the structure of HCN chnnels (Supplementry Fig. 10 nd d). This could point to n intrinsic flexiility of the C-linker region tht reflects its role in lignd-dependent gting 18. The quternry stte of the C-linker/CNBHD in the intct chnnel remins to e determined. 40 Ulens, C. & Siegelum, S.A., Regultion of hyperpolriztion-ctivted HCN chnnels y camp through gting switch in inding domin symmetry. Neuron 40 (5), (2003). 41 Zhou, Y., Moris-Crl, J.H., Kufmn, A., & McKinnon, R., Chemistry of ion coordintion nd hydrtion reveled y K+ chnnel-f complex t 2.0 A resolution. Nture 414 (6859), (2001). 42 Jing, Y. et l., Crystl structure nd mechnism of clcium-gted potssium chnnel. Nture 417 (6888), (2002). 43 Long, S.B., Cmpell, E.B., & Mckinnon, R., Crystl structure of mmmlin voltgedependent Shker fmily K+ chnnel. Science 309 (5736), (2005). 44 Arnold, K., Bordoli, L., Kopp, J., & Schwede, T., The SWISS-MODEL workspce: we-sed environment for protein structure homology modelling. Bioinformtics 22 (2), (2006). 12

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Jhn, Supplementry Figure S1 c Supplementry Figure S1, Solvent flttened, single-wvelength nomlous dispersion electron density mp (gry mesh) of the synptic SNARE complex with its two linker regions nd TMRs