Cryo-EM Structures of Calcium Homeostasis Modulator Channels In

bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Title: 2 Cryo-EM structures of calcium homeostasis modulator channels 3 in diverse oligomeric assemblies 4 5 One Sentence Summary: 6 Cryo-EM structures reveal the ATP conduction and oligomeric assembly 7 mechanisms of CALHM channels. 8 9 Authors: 10 Kanae Demura1,†, Tsukasa Kusakizako1,†, Wataru Shihoya1,†, Masahiro Hiraizumi1,2,†, 11 Kengo Nomura3, Hiroto Shimada1, Keitaro Yamashita1,4, Tomohiro Nishizawa1, Akiyuki 12 Taruno3, 5,*, Osamu Nureki1,* 13 14 Affiliations: 15 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 16 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; 2Discovery Technology Laboratories, 17 Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, 18 Aoba-ku, Yokohama 227-0033, Japan; 3Department of Molecular Cell Physiology, Kyoto 19 Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan; 20 4RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; 21 5Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 22 332-0012, Japan. 23 24 * To whom reprint requests should be addressed: [email protected] (A.T.), 25 [email protected] (O.N.). † These authors contributed equally to this work. 26

1 Abstract:

2 Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated

3 ATP-release channels involved in neural functions including neurotransmission in

4 gustation. Here we present the cryo-EM structures of killifish CALHM1, human

5 CALHM2, and C. elegans CLHM-1 at resolutions of 2.66, 3.51, and 3.60 Å, respectively.

6 The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction

7 site at the channel pore in the open state, and modulates the ATP conductance. The

8 CALHM2 undecamer and CLHM-1 nonomer structures show the different oligomeric

9 stoichiometries among CALHM homologs. We further report the cryo-EM structures of

10 the chimeric construct, revealing that the inter-subunit interactions at the transmembrane

11 domain define the oligomeric stoichiometry. These findings advance our understanding

12 of the ATP conduction and oligomerization mechanisms of CALHM channels. (121

13 words). 14

1 Main Text:

2 Introduction

3 Adenosine triphosphate (ATP) as an extracellular ligand plays essential roles in

4 various cellular functions, including neurotransmission (1), (2). Although anionic ATP

5 molecules cannot diffuse across the plasma membrane, ATP release from the cytosol is

6 mediated by ATP-permeable channels, such as connexin hemichannels, pannexin 1

7 (PANX1), volume-regulated anion channels (VRACs), and calcium homeostasis

8 modulator 1 (CALHM1) (3). CALHM1 was originally identified as a genetic risk factor

9 for late-onset Alzheimer’s disease (4), and recently identified as an essential component

10 of the neurotransmitter-release channel in taste bud cells that mediates the voltage-

11 dependent release of ATP to afferent gustatory neurons, whereby mediating the perception

12 of sweet, bitter, and umami tastes (5), (6), (7). CALHMs are regulated by the membrane

13 voltage and extracellular Ca2+. Strong de-polarization and extracellular Ca2+

2+ 14 concentration ([Ca ]o) reduction increase the open probability of CALHM channels (8),

15 (9). Six CALHM1 homologs were found in humans (10). CALHM genes are present

16 throughout vertebrates, but they have no sequence homology to other known genes.

17 CALHM2 modulates neural activity in the central nervous system (11). CALHM3

18 interacts with CALHM1, forming a CALHM1/CALHM3 heteromeric channel that

19 confers fast activation (12), (13). Outside of vertebrates, Caenorhabditis elegans (C.

20 elegans) possesses single CALHM ortholog, termed CLHM-1. The C. elegans CLHM-1

21 is expressed in sensory neurons and muscles, and functions as an ion channel (14), (15).

22 Many ATP-permeable channels have been structurally characterized (16), (17),

23 (18), (19), but the molecular mechanism of CALHM channels has remained unclear. Here

24 we report the cryo-EM structures of three CALHM homologs (CALHM1, CALHM2, and

1 C. elegans CLHM-1) and their chimeric construct in unexpectedly different oligomeric

2 states, revealing the oligomerization mechanism of CALHM channels. Furthermore, the

3 present cryo-EM structure of CALHM1 is the first to visualize the N-terminal helix,

4 which forms the constriction gate at the channel pore in the open state, and the

5 bioluminescence assay revealed that it modulates the ATP conductance. These findings

6 provide insights into the ATP conduction and oligomerization mechanisms of CALHM

7 channels.

9 Structure determination of CALHM1

10 For the structural analysis, we first screened the CALHM1 proteins from various

11 vertebrates by fluorescence detection gel filtration chromatography (FSEC) (20), and

12 identified the killifish Oryzias latipes CALHM1 (OlCALHM1) as a promising candidate

13 (Fig. S1A). OlCALHM1 and HsCALHM1 ('Hs' refers to Homo sapiens) share 62%

14 sequence identity and 78% sequence similarity within the transmembrane region. Next,

15 we analyzed the function of OlCALHM1. We transfected the DNA encoding the full-

16 length OlCALHM1 into HeLa cells, and measured the extracellular ATP concentration by

2+ 17 the luciferin–luciferase reaction (5) (Fig. S2A–C). Lowering [Ca ]o induced a larger

18 increase in the extracellular ATP concentration than in mock-transfected cells (Fig. S2A),

19 and ruthenium red (RUR), a known blocker of human and mouse CALHM1 channels,

20 abolished this ATP release (Fig. S2B), indicating that OlCALHM1 forms an ATP-release

2+ 21 channel activated by low [Ca ]o, as also reported for HsCALHM1 (5).

22 For the cryo-EM analysis, we expressed the CALHM protein in HEK293S GnT1-

23 cells. To facilitate the expression and purification of OlCALHM1, we truncated the

24 flexible C-termini starting from Trp296 (OlCALHM1EM). OlCALHM1EM retains the

2+ 1 ATP-release activity elicited by low [Ca ]o when expressed in HeLa cells (Fig. S2C).

2 Moreover, we confirmed that the purified OlCALHM1EM forms an ATP-permeable

3 channel by a liposome assay (Fig. S2D). The OlCALHM1EM protein was purified in GDN

4 micelles without Ca2+ and vitrified on a grid. The samples were imaged using a Titan

5 Krios electron microscope equipped with a K3 camera. The 2D class average showed

6 obvious C8 symmetry (Fig. S3A, B). The 3D map was reconstructed from 102,109

7 particles with the imposed C8 symmetry, at the 2.66 Å resolution based on the Fourier

8 shell correlation (FSC) gold standard criteria (Fig. 1A), which was sufficient for de novo

9 model building (Fig. S3C–E, Fig. S4A and Table S1). The processing summary is

10 provided in Fig. S3B. The N-terminal residues Met1 to Met6 and the C-termini after

11 Gln263 are disordered, consistent with the secondary structure prediction.

13 Structure of OlCALHM1

14 OlCALHM1EM forms an octamer with a length of 80 Å and a width of 110 Å (Fig.

15 1B, C). At the center of the octamer structure, a channel pore is formed along an axis

16 perpendicular to the membrane. The extracellular region projects approximately 15 Å

17 above the plasma membrane. The transmembrane region is 37 Å thick, and the

18 intracellular region protrudes about 28 Å from the plasma membrane. The subunit

19 consists of extracellular loops 1 and 2 (EL1: Phe37–Tyr47 and EL2: Phe128–Asp166),

20 an intracellular loop (IL: Arg83–Ala91), an N-terminal helix (NTH: Met7–Gln15), a

21 transmembrane domain (TMD: Gly25–Ser35, Tyr48–Leu70, Ile108–Ala127, and

22 Asn167–Cys205), and a C-terminal helix (CTH: Phe206–Arg260), with the C-terminus

23 on the cytoplasmic side (Fig. 1D, E). The TMD consists of four transmembrane helices

24 (TM1–4), which are aligned counterclockwise (Figs. 1D, E and S4B) as viewed from the

1 extracellular side. This TM arrangement is quite different from those in the connexin (16),

2 innexin (17), and LRRC8 (18), (19) structures (Fig. S4B). TM1 faces the channel pore,

3 while TM2 and TM4 face the phospholipid membrane. A clear density shows the N-

4 terminal helix (NTH) preceding TM1 and forming the channel pore, together with TM1

5 (Fig. S4A). TM3 is located between TMs 1, 2, and 4, connecting these structural elements.

6 TM4 is tilted by about 30° from the channel pore axis (perpendicular to the membrane

7 plane), and extends from the membrane lipid. Notably, TM2 is kinked at Asn71 (TM2a

8 and 2b), and TM3 is kinked at Leu107 and Gly121 (TM3a-c). Thereby, TM2b and TM3a

9 protrude into the intracellular region, and are stabilized by the hydrogen bonding

10 interactions of Asn71–Arg105 and Tyr97–Arg203. On the intracellular side, CTH adopts

11 a long helix and extends toward the adjacent subunit. The protruded TM2b and TM3a

12 form extensive van der Waals interactions with the CTH (Fig. 1F). On the extracellular

13 side, EL2 between TM3 and TM4 forms a short helix (EL2H) and wraps around the short

14 EL1 between TM1 and TM2, covering the extracellular side of the transmembrane region.

15 Between EL1 and EL2, two disulfide bonds are formed at the Cys41–Cys126 and Cys43–

16 Cys160 pairs, which stabilize the EL structure (Fig. 1G). The residues constituting the

17 transmembrane domain and the CTH, and the two cysteines in the extracellular loops are

18 highly conserved in the CALHM1 orthologs, including HsCALHM1 (Fig. S1A–C).

19 A previous study reported that two cysteines in mouse CALHM1 are

20 palmitoylated and associated with the post-translational regulation of the channel gating

21 and localization (21). These two cysteines are completely conserved in the CALHM1

22 orthologs (Fig. S1A). In the OlCALHM1EM structure, the corresponding cysteines

23 Cys100 and Cys205 are located at TM3a and the intracellular end of TM4, respectively

24 (Fig. 1F). Although we could not find the density of the palmitoylation in our EM map,

1 these residues face the lipid bilayer and are likely to be palmitoylated.

2 The P86L polymorphism in HsCALHM1 is associated with Alzheimer’s disease

3 (4). In the OlCALHM1EM structure, the corresponding residue Thr85 is located on the

4 intracellular loop between TM2b and TM3a. Therefore, Thr85 is not oriented toward the

5 channel pore and not directly associated with the TMD-CTH interface. Given that the

6 palmitoylation site is also located on TM2b (Fig. 1F), the protruded TM2b and TM3a

7 probably have an important role in channel function.

8 In HsCALHM1, the negative charge of Asp121 reportedly plays a critical role in

9 ion permeation property (8). This Asp residue is highly conserved in the CALHM1

10 orthologs, and the corresponding Asp120 in OlCALHM1 forms a salt bridge with Lys122

11 in TM3 (Fig. 1H), stabilizing its bent conformation at Gly121. Moreover, Asp120 forms

12 a hydrogen bond with Tyr34 in TM1 and an electrostatic interaction with Lys178 in TM4.

13 These residues are also conserved in HsCALHM1. Overall, Asp120 contributes to the

14 structural organization of the TM helices inside the channel.

16 Subunit interface in OlCALHM1

17 The subunit interface is formed at the transmembrane and intracellular regions

18 (Fig. 2A). Unlike connexin (16), innexin (17), and LRRC8 (18), (19), the extracellular

19 region is not involved in the subunit interface. At the transmembrane region (Fig. 2B),

20 TM2 forms extensive hydrophobic interactions with TM4’ (apostrophe indicates the

21 adjacent subunit). At the extracellular and intracellular ends of the interface, Tyr51 and

22 Asn72 in TM2 form hydrogen bonds with Gln182 and Arg200 in TM4’, respectively (Fig.

23 2B). EL1 also forms hydrogen-bonding interactions with TM4’ (Fig. 2C). Moreover, the

24 NTH regions interact with each other between adjacent subunits: Gln12 in the NTH forms

1 a hydrogen bonding interaction with Ser13 in the NTH’ (Fig. 2C). At the intracellular

2 region, the long CTH extends to the adjacent subunit and forms extensive interactions

3 with it (Fig. 2D). Furthermore, Glu254 forms a salt bridge with Lys213 in the CTH

4 between the two adjacent subunits (Fig. 2D).

5 In addition, there are extra densities at the subunit interface (Figs. 2E, F and S4A).

6 Based on their characteristic shape, we assigned them to the cholesteryl hemisuccinate

7 (CHS) molecules used for solubilization (CHS1, 2) (Fig. 2E). These CHS molecules are

8 surrounded by TM1 (Ser36) and TM1’ (Met28 and Leu19), TM3 (Val116), and TM4

9 (Arg200 and Leu189), and bridge the gap between the subunits behind the NTH,

10 stabilizing the subunit interface and pore architecture. A lipid-mediated subunit interface

11 has not been observed in connexin26, LRRC8, or other gap junction channels, and is a

12 unique structural feature of OlCALHM1. Moreover, two CHS molecules are observed on

13 the membrane side (CHS3, 4) (Fig. 2F). These CHS molecules form extensive

14 hydrophobic interactions with the channel and fill the cleft between TMs1, 3, and 4. The

15 residues involved in the interaction with CHS are highly conserved in the CALHM1

16 orthologs (Fig. S1), suggesting that lipids play an important role in the structural

17 stabilization of the CALHM1 channels.

19 Pore architecture of OlCALHM1

20 OlCALHM1EM has a channel pore along a central axis perpendicular to the

21 membrane. In the structures of gap junction channels (connexin and innexin) (16), (17),

22 the NTH and extracellular loop form two constriction sites in the pore. In the

23 OlCALHM1EM structure, only the NTH forms the single constriction site in the pore (Fig.

24 3A). At the constriction site, the neutral residues Gln9, Gln12, and Ser13 face the pore,

1 with the narrowest diameter of 15.7 Å at Gln9 (Fig. 3B). Previous optical and

2 electrophysiological analyses of the permeation of fluorescence dyes and ions with

3 different sizes suggested that HsCALHM1 has a large pore greater than 14 Å (10), which

4 is consistent with the diameter at the narrowest neck of OlCALHM1EM, allowing the

5 permeation of all hydrated ions and ATP (~7 Å). HsCALHM1 is both cation and anion

6 permeable at PNa: PK: PCa: PCl = 1: 1.14: 13.8: 0.52 (8), (10). The pore-facing residues in

7 the NTH are neutral (Gln9, Gln12, and Ser13), accounting for the weak charge selectivity.

8 Taken together, the pore architecture in the OLCALHM1EM structure is in excellent

9 agreement with the previous functional analysis of HsCALHM1.

10 To elucidate the functional role of the NTH, we truncated the NTH of OlCALHM1

11 (ΔN11 and ΔN19) (Fig. 3C) and measured the ATP release activity by a cell-based assay.

12 In the Ca2+-free open state, the ATP release activities of the NTH-truncated mutant

13 channels were markedly enhanced as compared to the wild-type, indicating that the NTH

14 importantly functions in the ATP release (Fig. 3D). However, in the Ca2+-bound closed

15 state, the NTH-truncated mutants showed little ATP release activities (Fig. 3E). These

16 observations indicate that the NTH observed in our CALHM1 structure is indeed involved

17 in the channel conduction of ATP; however, the other structural components are

18 associated with the Ca2+-dependent channel closing.

20 Structures of HsCALHM2 and CeCLHM-1 and structural differences

21 among CALHM homologs

22 Next, we investigated the functional and structural diversity of the CALHM

23 family. Although various members of the CALHM family have been identified (11), (14),

24 (15), their ATP conductance activities remain elusive. Thus, we measured the ATP

1 release activities of HsCALHM2 and CeCLHM-1 by a cell-based assay. HsCALHM2-

2 and CeCLHM-1-transfected HeLa cells exhibited greater RuR-sensitive increases in the

2+ 3 extracellular ATP concentration in response to lowering [Ca ]o compared to mock-

4 transfected cells, indicating that HsCALHM2 and CeCLHM-1 form ATP-release

2+ 5 channels activated by low [Ca ]o, as in the CALHM1 channels (Fig. S2A, B). To further

6 understand the ATP conduction mechanisms of the CALHM family members, we

7 performed the cryo-EM analysis of HsCALHM2 and CeCLHM-1. HsCALHM2 and

8 CeCLHM-1 were purified without Ca2+ and exchanged into the amphipol PMAL-C8. The

9 cryo-EM images were acquired on a Titan Krios electron microscope equipped with a

10 Falcon 3 direct electron detector. The 2D class averages of HsCALHM2 and CeCLHM-

11 1 showed the top views of 11-mer and 9-mer, respectively. We finally reconstructed the

12 EM density maps of HsCALHM2 and CeCLHM-1 at resolutions of 3.51 Å and 3.6 Å,

13 respectively, according to the gold-standard Fourier shell correlation (FSC) = 0.143

14 criteria (Figs. S5, S6 and Table S1).

15 Unlike the OlCALHM1 8-mer structure, HsCALHM2 and CeCLHM-1 form the

16 11-mer (approximate dimensions of 93 Å in height × 143 Å in intracellular width) and

17 the 9-mer (approximate dimensions of 93 Å in height ×126 Å in intracellular width),

18 respectively (Fig. 4A). The 11-mer structure of HsCALHM2 is similar to that of the

19 recently reported structure in the open state (22). Each subunit of the three homologs,

20 OlCALHM1, HsCALHM2, and CeCLHM-1, adopts almost similar architectures

21 composed of the transmembrane domain (TMD) and the C-terminal domain (CTD) (Fig.

22 4B, C). At the TMD, both HsCALHM2 and CeCLHM-1 have the 4-TM helix topology,

23 in which the NTH and TM1 of HsCALHM2 and CeCLHM-1 are also expected to face

24 the pore, similar to OlCALHM1. However, the NTH and TM1 are not visualized in the

1 density maps of HsCALHM2 and CeCLHM-1, indicating the conformational flexibility

2 of these regions. At the extracellular regions, two disulfide bonds between extracellular

3 loops 1 (EL1) and 2 (EL2) are conserved among the three CALHM homologs (Fig. S7).

4 EL2, between TM3 and TM4, forms short helices that are arranged differently in the three

5 structures. At the CTD, the three CALHM homologs adopt distinct architectures (Fig. 4B,

6 C). In contrast to OlCALHM1, HsCALHM2 and CeCLHM-1 have a CTH and additional

7 helices, and their arrangements are different between the two homologs (Fig. 4B, C): The

8 CTD of HsCALHM2 consists of a CTH (Arg215–Phe250), which turns toward the pore

9 at Phe251, and three short helices. The CTD of CeCLHM-1 consists of a CTH (Leu220–

10 Arg254), which turns toward the membrane at Phe257, and at least one short helix. In

11 addition to these structural observations, the low sequence identities within the CTDs

12 among the three homologs (OlCALHM1 vs. HsCALHM2: 23%, OlCALHM1 vs.

13 CeCLHM-1: 32%) suggest the structural divergence of the CTD among the CALHM

14 family members.

15 Similar to OlCALHM1, both the TMD and CTD are involved in the subunit

16 interactions in HsCALHM2 and CeCLHM-1 (Fig. 4D–F). At the TMD, TM2 of one

17 subunit and TM4’ of the neighboring subunit form extensive hydrophobic interactions

18 (Fig. 4D, E). Particularly, two Pro residues on TM2 and TM3, and two Trp residues on

19 TM3 and TM4’ form van der Waals interactions, resulting in the tight contacts of the

20 three TM helices (TM2, TM3, and TM4’), which are conserved among the three CALHM

21 structures (Figs. 4D, E and S7). Structural comparisons of the three CALHM homologs

22 show the different relative positions and orientations of TM2 relative to TM4’ of the

23 neighboring subunits (Fig. 4D). The subunit interfaces between TM2 and TM4’ in

24 OlCALHM1 are formed by tighter hydrophobic interactions, relative to those in

1 HsCALHM2 and CeCLHM-1: The distances between the Cα atoms of the intracellular

2 ends in TM2 and TM4’ in OlCALHM1, HsCALHM2, and CeCLHM-1 are 14.4, 17.9,

3 and 16.8 Å, respectively (Fig. 4D). These subtle differences in the subunit interfaces at

4 the TMD are likely to be associated with the different oligomeric assemblies. At the CTD

5 in the three CALHM structures, the neighboring CTHs entangle together and form

6 stacking interactions between the conserved aromatic residues: Phe and His residues in

7 one subunit, a Phe residue in the adjacent subunit, and two Tyr residues from the adjacent

8 subunit on the opposite side (Figs. 4F and S7). Taken together, the structural comparisons

9 among the three CALHM homologs reveal both structural conservation (TM topology)

10 and divergence (structures of CTD and oligomeric assembly).

12 Oligomeric assembly mechanism of CALHM channels

13 The three different oligomeric assembly structures of the CALHM homologs

14 probably arise from the slight variations in the inter-subunit interactions formed at the

15 TMD and CTD. To investigate whether the TMD or CTD defines the oligomeric

16 stoichiometry, we constructed a chimeric channel, consisting of the TMD from

17 OlCALHM1 and the CTD from HsCALHM2 (named ‘OlCALHM1c’). The purified

18 OlCALHM1c was exchanged into amphipol PMAL-C8 and subjected to the cryo-EM

19 analysis. Without imposing any symmetry, the 3D classification provided cryo-EM maps

20 of two different oligomeric states, the 8-mer and the 9-mer, but no 11-mer structure was

21 obtained (Figs. 5A, S8). We finally determined the cryo-EM structures of the 8-mer and

22 the 9-mer, both at resolutions of 3.4 Å (Figs. 5A, B, S8 and Table S1). The dimensions of

23 the 8-mer and 9-mer structures of OlCALHM1c are consistent with the 8-mer and 9-mer

24 structures of OlCALHM1 and CeCLHM-1, respectively (Fig. 5B). Since the densities of

1 the CTDs are obscure in both the 8-mer and 9-mer EM maps (Fig. 5A), we did not model

2 the entire CTD of the 8-mer and the side chains at the CTD of the 9-mer (Fig. 5B). By

3 contrast, the EM density maps corresponding to the TMD are clearly resolved in both

4 structures, allowing the identification of the residues in the TMD (Fig. 5B). The TMD

5 structures of the respective subunits are almost identical between the 8-mer and the 9-mer

6 (RMSD of 0.55 Å over 184 Cα atoms) (Fig. 5C).

7 The subunit interfaces at the TMD of OlCALHM1c are almost the same as those

8 of OlCALHM1EM, formed by hydrophobic interactions between TM2 of one subunit and

9 TM4’ (Fig. 5D). Consistently, the 8-mer structure can be well superimposed with

10 OlCALHM1EM, with the same TMD element. On the other hand, superimposition of the

11 8-mer and the 9-mer based on a subunit shows a 0.6 Å displacement of the Cα atoms of

12 Ile202 on the intracellular end of TM4’ (Fig. 5D). The substitution of the CTD probably

13 allowed this subtle difference of TM4’ at the TMD subunit interfaces, resulting in the 9-

14 mer assembly (Fig. 5B, E). These observations indicate that the assembly stoichiometry

15 is mainly defined by the subunit interactions at the TMD (e.g., the TMD of OlCALHM1

16 forms the 8-mer or the 9-mer). This notion suggests that the diverse oligomeric states in

17 CALHM channels are generally determined by the subunit interactions at the TMD.

18 Although the three CALHM homologs adopt similar TM topologies, the TMD of

19 OlCALHM1 has low sequence identities with those of HsCALHM2 (29%) and

20 CeCLHM-1 (23%). These sequence differences within the TMDs among the CALHM

21 family members are likely to be reflected in their diverse oligomeric assemblies and

22 eventually in the different diameters of the channel pore, which may be associated with

23 the different specificities of the released substrates at distinct expression sites (5), (11),

24 (14).

2 Discussion

3 In this study, we present the cryo-EM structures of three CALHM homologs and

4 the two oligomeric states of the chimeric construct, revealing the ATP conduction and

5 oligomeric assembly mechanisms. Notably, the present 2.66 Å-resolution OlCALHM1

6 structure allowed the accurate modeling of the NTH and TM1 (Fig. S4A), which form the

7 pore architecture in the Ca2+-free open state (Fig. 3A, B). The analysis of the ΔNTH

8 mutants indicates that the NTH is involved in the channel conduction of the ATP (Fig.

9 3D). Although the NTH and TM1 are disordered in our HsCALHM2 and CeCLHM-1

10 structures, the comparable arrangement of the TM helices among OlCALHM1,

11 HsCALHM2, and CeCLHM-1 suggests that the NTH would similarly constitute the

12 channel pores in the CALHM family members. The pore constriction by the NTH is also

13 observed in gap junction channels (16), (17), indicating that this is a conserved structural

14 feature of these oligomeric channels, despite the lack of sequence similarity among them.

15 The ΔNTH mutant showed almost no ATP-release activity upon Ca2+-binding,

16 similar to the wild-type (Fig. 3E), suggesting that other structural components are

17 associated with the channel gating in OlCALHM1 (Fig. 6A, B). In the OlALHM1EM

18 structure, TM1 protrudes towards the channel pore, as compared to the other TMs (Fig.

19 1D). Recently, the cryo-EM structures of HsCALHM2 were reported in the open and

20 inhibited states at 3.3 and 2.7 Å resolutions, respectively (22). These structures showed

21 channel pore closing by the swing motion of TM1 by about 60° towards the pore axis,

22 driven by the binding of the inhibitor RUR at the base of the TM segment. Therefore,

23 these structures suggest the large rearrangement of TM1 upon the channel gating (22).

24 Given the structural and sequence similarities between OlCALHM1 and HsCALHM2

1 (Figs. 4B, C and S7), TM1 may undergo a large structural movement and block the pore

2 upon Ca2+ binding in OlCALHM1 (Fig. 6A, B).

3 For the subunit assembly of CALHM family proteins, the interactions at the TMD

4 are mostly important for the oligomeric stoichiometry. The hydrophobic residues

5 involved in the subunit interactions between TM2–TM4’ at the TMD are conserved in the

6 CALHM1 orthologs, except for Met187 and Phe194 (Fig. S1A–C), implying a similar

7 assembly number of HsCALHM1. In taste bud cells, CALHM1 and CALHM3 form a

8 functionally heteromeric channel with fast activation (12). HsCALHM1 and HsCALHM3

9 have sequence identities of 47% overall and 54% within the TMD (Fig. S9), which are

10 relatively higher as compared to the sequence identity between HsCALHM1 and

11 HsCALHM2 (30% over the full-length and 33% within the TMD). Moreover, the residues

12 that are probably involved in the subunit assembly are conserved in both HsCALHM1

13 and HsCALHM3, implying that they can form a heterooctamer, although the different

14 residues of the CTD could slightly modify this assembly. Overall, our current study

15 provides the ATP conduction and diverse assembly mechanisms of the CALHM family

16 proteins.

19 References and Notes: 20 1. G. Burnstock, Historical review: ATP as a neurotransmitter. Trends in 21 Pharmacological Sciences. 27, 166–176 (2006). 22 2. M. P. Abbracchio, G. Burnstock, A. Verkhratsky, H. Zimmermann, Purinergic 23 signalling in the nervous system: an overview. Trends in Neurosciences. 32, 19– 24 29 (2009). 25 3. A. Taruno, ATP release channels. International Journal of Molecular Sciences. 26 19, 808 (2018). 27 4. U. Dreses-Werringloer, J. C. Lambert, V. Vingtdeux, H. Zhao, H. Vais, A. 15

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12 Acknowledgements: We thank T. Nakane for assistance with the single-particle

13 analysis. We also thank the staff scientists at the University of Tokyo’s cryo-EM facility,

14 especially K. Kobayashi, H. Yanagisawa, A. Tsutsumi, M. Kikkawa, and R. Danev.

15 Funding: This work was supported by a MEXT Grant-in-Aid for Specially Promoted

16 Research (grant 16H06294) to O.N, and JST PRESTO (grant JPMJPR1886) to A.T.

17 Authors’ contributions: K.D. prepared the cryo-EM sample of OlCALHM1 and

18 performed the liposome assay. K.D. and T.K. collected and processed the cryo-EM data,

19 and built the structure of OlCALHM1. T.K. prepared the cryo-EM sample of the CALHM

20 chimera, collected and processed the cryo-EM data, and built the structures of

21 HsCALHM2, CeCLHM-1, and the CALHM chimera. W.S. initially screened the

22 CALHM homologs, established the expression and purification procedure, and prepared

23 the cryo-EM sample of HsCALHM2. M.H. screened and prepared the cryo-EM sample

24 of CeCLHM-1, collected and processed the cryo-EM data, and built the structure of

25 CeCLHM-1. T.N. assisted with the data collection and processing. K.Y. assisted with the

26 data processing and structure refinement. K.N. and A.T. measured the ATP-release

27 activities of the CALHM proteins. K.D., T.K., W.S., and O.N. mainly wrote the

1 manuscript. A.T. and O.N. supervised the research.

2 Competing interests: M.H. is a graduate student at Mitsubishi Tanabe Pharma

3 Corporation and is supported by the company with non-research funds. The company has

4 no financial or other interest in this research.

5 Data and materials availability:

7 Figure legends:

8 Fig. 1: Overall structure of OlCALHM1.

9 (A) Local resolution of the OlCALHM1EM structure, estimated by RELION. (B and C)

10 Overall structure of the OlCALHM1EM octamer, viewed parallel to the membrane (B) and

11 from the extracellular side (C). (D) The subunit structure of OlCALHM1EM, viewed

12 parallel to the membrane. Each region is colored as follows: NTH, pink; TM1, purple;

13 EL1, white; TM2a, light blue; TM2b, IL1, and TM3a, cyan; TM3b and 3c, light green;

14 EL2, yellow; TM4, orange; CTH, red. The N- and C-termini are indicated by ‘N’ and ‘C’,

15 respectively. (E) Schematic representation of the OlCALHM1 membrane topology,

16 colored as in Fig. 1D. (F–H) Close-up views of the intracellular side (F), the two disulfide

17 bonds in the extracellular region (G), and the interactions between TM1 and the other

18 TMs around Asp120 (H).

19 20 Fig. 2: Subunit interfaces between two adjacent subunits. 21 (A) Overview between two adjacent subunits, viewed from the extracellular side. (B–D)

22 Close-up views between two adjacent subunits at the transmembrane region (B and C)

23 and intracellular region (D). (E and F) Close-up views of CHS1, 2 (E) and CHS3, 4 (F).

24 CHS molecules are indicated by grey stick models.

1 2 Fig. 3: Pore architecture and ATP conduction of OlCALHM1.

3 (A) Cross-sections of the surface representation for the OlCALHM1EM subunits, showing

4 the ion channel pore. (B) The pore radius for the OlCALHM1EM structure along the pore

5 center axis. The pore size was calculated with the program HOLE (23). (C) Design of the

6 ΔNTH constructs. The residues in the NTH and the loop between NTH and TM1 are

7 shown as stick models. (D and E) Time courses of extracellular ATP levels due to release

8 from HeLa cells transfected with the empty vector (mock), WT OlCALHM1, or NTH-

9 truncated (ΔN11 or ΔN19) OlCALHM1 following exposure to essentially zero (17 nM)

2+ 10 (D) or normal (1.9 mM) [Ca ]o (E). Data are displayed as means ± S.E.M. n = 15 (Mock),

11 n = 16 (WT, ΔN11, and ΔN19) in D. n = 16 in each group in E.

12 13 Fig. 4: Structural differences among OlCALHM1, HsCALHM2, and 14 CeCLHM-1. 15 (A) Structural comparisons among OlCALH1, HsCALHM2, and CeCLHM1. Overall

16 structures are viewed from the extracellular side (top) and the intracellular side (bottom),

17 colored as in Fig. 1D. (B) Subunit structures of the three CALHM channels, colored as in

18 Fig. 1D. Adjacent subunits are shown in semi-transparent colors. (C) Superimposition of

19 the subunit structures of the three CALHM channels, viewed from the membrane side

20 (left) and the intracellular side (right). OlCALHM1, HsCALHM2, and CeCLHM-1 are

21 colored purple, cyan, and yellow, respectively. (D) Close-up view of the superimposition

22 of the subunit structures of the three CALHM channels, colored as in Fig. 4C. Red arrows

23 indicate the different orientations of TM4’ in the adjacent subunits among the three

24 CALHM structures. (E) Subunit interfaces at the TMD of the three CALHM channels.

25 TM2, TM3, and TM4 are colored blue, green, and orange, respectively. The conserved

1 residues among the CALHM channels are shown in stick models and semi-transparent

2 CPK representations. Other residues involved in the interactions between TM2 and TM4’

3 in the adjacent subunit are shown in stick models. (F) Subunit interfaces at the CTDs of

4 the three CALHM channels. The center CTDs are colored red, and the two adjacent CTDs

5 are colored purple and green. The conserved residues among the CALHM channels are

6 shown in stick models and semi-transparent CPK representations. Other residues

7 involved in interactions between adjacent CTDs are shown in stick models. Hydrogen

8 bonds and salt bridges are shown as black dashed lines.

9 10 Fig. 5: Structures and oligomerization states of the chimeric CALHM 11 proteins. 12 (A) EM density maps of the OlCALHM1c 8-mer (left) and 9-mer (right), colored

13 according to the local resolution (blue: high resolution to red: low resolution), estimated

14 by RELION. (B) Overall structures of the OlCALHM1c 8-mer (left) and 9-mer (right),

15 viewed from the membrane side, colored as in Fig. 1D. (C) Superimposition of the

16 OlCALHM1c 8-mer and 9-mer, viewed from the membrane side. The 8-mer and 9-mer

17 structures are colored pale blue and pale pink, respectively. (D) Close-up view of the

18 superimposition of the OlCALHM1c 8-mer and 9-mer, colored as in Fig. 5C. The

19 conserved residues among the CALHM channels are shown in stick models and semi-

20 transparent CPK representations. The red arrow indicates the different orientations of

21 TM4’ in the adjacent subunit between the 8-mer and 9-mer structures. (E)

22 Superimposition of the OlCALHM1c 8-mer and 9-mer, viewed from the extracellular side,

23 colored as in Fig. 5C. The two structures are superimposed based on TM2 of subunit 1.

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Fig. 6: Insight into channel gating

2 (A and B) Schematic model of the Ca2+-dependent gating mechanism. In the Ca2+-free

3 open state, TM1 forms the stable interaction with TM3 and moves perpendicularly to the

4 membrane. The NTH forms the constriction site in the pore (A). In the Ca2+-bound closed

5 state, TM1 blocks the pore (B).

1 Supplementary Materials:

3 Materials and Methods:

4 Expression and purification of OlCALHM1, HsCALHM2, and the

5 chimeric construct OlCALHM1c

6 The DNAs encoding the Oryzias latipes CALHM1 channel (UniProt ID:

7 H2MCM1) and the Homo sapiens CALHM2 (UniProt ID: Q9HA72) were codon

8 optimized for eukaryotic expression systems, synthesized (Genewiz, Inc.), and served as

9 the templates for subcloning. To improve the expression and thermostability, the C-

10 terminal residues starting from Trp296 were truncated (OlCALHM1EM). The chimeric

11 construct consists of the TMD in OlCALHM1 (Met1–Ala209) followed by the CTD in

12 HsCALHM2 (Ser213–Ser323). These DNA fragments were ligated into a modified pEG

13 BacMam vector, with a C-terminal green fluorescent protein (GFP)-His8 tag and a

14 tobacco etch virus (TEV) cleavage site, and were expressed in HEK293S GnTI− (N-

15 acetylglucosaminyl-transferase I-negative) cells (American Type Culture Collection,

16 catalog no. CRL-3022) (24). Cells were collected by centrifugation (8,000 × g, 10 min,

17 4°C). For the cells expressing OlCALHM1c, the cells were disrupted by probe sonication

18 and the membrane fraction was collected by ultracentrifugation (186,000 × g, 1 h, 4°C).

19 The cells or the membrane fraction were solubilized for 1 h in buffer containing 50 mM

20 Tris, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol (DTT), 1% DDM (Calbiochem), and

21 0.2% cholesterol hemisuccinate (CHS). The mixture was centrifuged at 40,000g for 20

22 min, and the supernatant was incubated with CNBr-Activated Sepharose 4 Fast Flow

23 beads (GE Healthcare) coupled with an anti-GFP nanobody (GFP enhancer or GFP

24 minimizer) (25) for 2 h at 4°C. The protein-bound resins were washed with gel filtration

1 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol) containing 0.05% GDN

2 for OlCALHM1EM and OlCALHM1c or 0.06% digitonin for HsCALHM2, and further

3 incubated overnight with TEV protease. The flow through was concentrated using a

4 centrifugal filter unit (Merck Millipore, 100 kDa molecular weight cutoff), and separated

5 on a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with gel

6 filtration buffer. The peak fractions of the protein were pooled.

8 Expression and purification of CeCLHM-1

9 The Caenorhabditis elegans calcium homeostasis modulator protein (CeCLHM-

10 1, Uniprot: Q18593) cDNA was synthesized, codon-optimized for expression in human

11 cell lines, and cloned into the pcDNA3.4 vector. The CeCLHM-1 sequence was fused

12 with a C-terminal His8 tag and enhanced green fluorescent protein (EGFP), followed by

13 a tobacco etch mosaic virus (TEV) protease site. HEK293S GnTI− cells were transiently

14 transfected at a density of 2.0 × 106 cells ml−1, with the plasmids and FectoPRO (Polyplus).

15 Approximately 360 μg of the CeCLHM-1 plasmid were premixed with 360 μL FectoPRO

16 reagent in 80 mL of fresh FreeStyle 293 medium, for 10–20 min before transfection. For

17 transfection, 80 ml of the mixture was added to 0.8 L of cell culture and incubated at 37°C

18 in the presence of 8% CO2. After 18–20 h, 2.2 mM valproic acid was added, and the cells

19 were further incubated at 30°C in the presence of 8% CO2 for 48 h. The cells were

20 collected by centrifugation (3,000 × g, 10 min, 4°C) and disrupted by Dounce

21 homogenization in hypotonic buffer (50 mM HEPES-NaOH (pH 7.5), 10 mM KCl, 0.04

22 mg ml−1 DNase I, and protease inhibitor cocktail). The membrane fraction was collected

23 by ultracentrifugation (138,000 × g, 1 h, 4°C), and solubilized for 1 h at 4°C in buffer

24 (50 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, 1.5% (w/v) N-dodecyl β-D-maltoside

1 (DDM), and 0.15% (w/v) cholesterol hemisuccinate (CHS)). After ultracentrifugation

2 (138,000 × g, 30 min, 4°C), the supernatant was incubated with AffiGel 10 (Bio-Rad)

3 coupled with a GFP-binding nanobody, for 2 h at 4°C. The resin was washed five times

4 with 3 CVs of wash buffer (50 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, and 0.06%

5 glyco-diogenin (GDN)), and gently suspended overnight with TEV protease to cleave the

6 His8-EGFP tag. After the TEV protease digestion, the flow-through was pooled,

7 concentrated, and purified by size-exclusion chromatography on a Superose 6 Increase

8 10/300 GL column (GE Healthcare), equilibrated with SEC buffer (20 mM HEPES-

9 NaOH (pH 7.5), 150 mM NaCl, and 0.06% GDN). The peak fractions of the protein were

10 pooled.

11 12 Grid preparation

−1 13 The purified OlCALHM1EM protein in GDN was concentrated to 5 mg ml . For

14 cryo-EM analyses of HsCALHM2, CeCLHM-1, and OlCALHM1c, the detergents were

15 exchanged with amphipols. The purified HsCALHM2 in digitonin, CeCLHM-1 in GDN,

16 and OlCALHM1c in GDN were mixed with amphipol PMAL-C8 (Anatrace) at a 1:100

17 ratio (w/w) for 2–2.5 h at 4°C. Detergents were removed using Bio-Beads SM-2 (Bio-

18 Rad, 100 mg per 1 ml mixture) for 2–2.5 h at 4°C. After removal of the Bio-Beads, the

19 samples were concentrated and subjected to size-exclusion chromatography on a

20 Superose 6 Increase 10/300 GL column (GE Healthcare), equilibrated with buffer (20

21 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol). The peak fractions containing

22 HsCALHM2, CeCLHM-1, and the OlCALHM1c in PMAL-C8 were concentrated to 1.6,

23 2.2, and 1 mg ml−1, respectively. Portions (3 µl) of the concentrated samples were applied

24 to glow-discharged R1.2/1.3 Cu/Rh 300 mesh grids (Quantifoil). The grids were

1 subsequently blotted (4 s for OlCALHM1EM, 8 s for HsCALHM2, 4 s for CeCLHM-1,

2 and 4 s for OlCALHM1c) and vitrified using a Vitrobot Mark IV (FEI) under 4°C and

3 100% humidity conditions.

4 5 EM image acquisition and data processing

6 For OlCALHM1EM and OlCALHM1c, data collections were performed on an FEI

7 Titan Krios (FEI) electron microscope, operating at an acceleration voltage of 300 kV and

8 equipped with a BioQuantum K3 imaging filter and a K3 direct electron detector (Gatan).

9 EM images were acquired at a nominal magnification of 105,000 ×, corresponding to a

10 physical pixel size of 0.83 Å, using the SerialEM software (26). For OlCALHM1EM,

11 movies were dose fractionated to 54 frames at a dose rate of 14.9 e− pixel−1 per second,

12 resulting in a total accumulated dose of 59.4 e− Å−2. For OlCALHM1c, movies were dose

13 fractionated to 48 frames at a dose rate of 14.0 e− pixel−1 per second, resulting in a total

14 accumulated dose of 50 e− Å−2. For HsCALHM2 and CeCLHM-1, data collections were

15 performed on a 300 kV Titan Krios electron microscope equipped with a Falcon III direct

16 electron detector. EM images were acquired at a nominal magnification of 96,000 ×,

17 corresponding to a calibrated pixel size of 0.8346 Å pixel−1, using the EPU software. For

18 HsCALHM2, movies were dose fractionated to 60 frames at a dose rate of 0.95 e− pixel−1

19 per second, resulting in a total accumulated dose of 60 e− Å−2. For CeCLHM-1, movies

20 were dose fractionated to 48 frames at a dose rate of 0.7 e− pixel−1 per second, resulting

21 in a total accumulated dose of 50 e− Å−2. For all datasets, the movie frames were aligned

22 in 4 × 4 patches and dose-weighted using RELION (27). CTF estimation was performed

23 by CTFFIND 4.1 (28).

24 For the OlCALHM1EM dataset, a total of 1,231,992 particles were extracted from

1 3,075 micrographs in 3.55 Å pixel−1 using AutoPick in RELION-3.0 (27), applying the

2 two-dimensional class averages of reference-free auto-picked particles based on a

3 Laplacian-of-Gaussian (LoG) filter as templates. The initial model was generated in

4 RELION. The particles were passed to RELION to calculate the two-dimensional

5 classification, refinement, and post-processing. Since some classes of the two-

6 dimensional classification exhibited the density for eight subunits, 137,146 particles in 3

7 good classes were re-extracted in the original pixel size of 0.83 Å pixel−1 and refined in

8 C8 symmetry. The particles were further cleaned using 3D classification, and 102,109

9 good particles were subsequently subjected to micelle subtraction, polishing (29), and

10 CTF refinement. The overall gold-standard resolution calculated at the Fourier shell

11 correlation (FSC = 0.143) (30) was 2.66 Å.

12 For the HsCALHM2 dataset, a total of 946,353 particles were initially extracted

13 from 2,110 micrographs, with a binned pixel size of 3.52 Å pixel−1, as described above.

14 These particles were subjected to several rounds of 2D and 3D classifications, resulting

15 in the best class from the 3D classification, which contained 365,116 particles. These

16 particles were then re-extracted with 1.40 Å pixel−1, and subsequently subjected to 3D

17 refinement with C11 symmetry, polishing, CTF refinement, and micelle subtraction. The

18 final 3D refinement with C11 symmetry and postprocessing yielded a density map with

19 an overall resolution of 3.51 Å at a gold-standard FSC = 0.143. The image processing

20 workflow is described in Fig. S5.

21 For the CeCLHM-1 dataset, a total of 832,750 particles were initially extracted

22 from 2,906 micrographs, with a binned pixel size of 3.01 Å pixel−1, as described above.

23 These particles were subjected to several rounds of 2D classification, resulting in the best

24 classes containing 261,169 particles. These particles were re-extracted with 1.5 Å pixel−1,

1 and subjected to 3D classification, resulting in the best classes containing 65,887 particles.

2 The particles were subsequently subjected to 3D refinement with C9 symmetry, polishing,

3 and CTF refinement. The final 3D refinement with C9 symmetry and postprocessing

4 yielded a density map with an overall resolution of 3.6 Å at a gold-standard FSC = 0.143.

5 The image processing workflow is described in Fig. S6.

6 For the OlCALHM1c dataset, a total of 1,689,949 particles were initially extracted

7 from 2,808 micrographs, with a binned pixel size of 3.39 Å pixel−1, as described above.

8 These particles were subjected to several rounds of 2D and 3D classifications, resulting

9 in the two distinct classes from the 3D classification, which represented the 8-mer and 9-

10 mer structures. The 8-mer and 9-mer classes contained 233,566 and 227,781 particles,

11 respectively. These particles were separately re-extracted with 1.41 Å pixel−1, and

12 subsequently subjected to 3D refinement with C8 or C9 symmetry, respectively. The final

13 3D refinement and postprocessing of the two classes yielded density maps with overall

14 resolutions of 3.4 Å in both cases, at a gold-standard FSC = 0.143. The image processing

15 workflow is described in Fig. S8.

16 17 Model building and refinement

18 The model of OlCALHM1EM was built de novo into the density map using COOT

19 (31) and rebuilt using Rosetta (32). The model was again rebuilt using COOT with

20 multiple rounds of real-space refinement in PHENIX (33), (34), with secondary structure

21 restraints. The models of HsCALHM2 and CeCLHM-1 were built using COOT with

22 multiple rounds of real-space refinement in PHENIX, with secondary structure restraints.

23 For the OlCALHM1c 8-mer and 9-mer, the TMD of OlCALHM1EM were modeled into

24 the density map by rigid-body fitting and modified using COOT with multiple rounds of

1 real-space refinement in PHENIX, with secondary structure restraints.

2 For cross-validation, the final models were randomly shaken and refined against

3 one of the half maps generated in RELION. Fourier shell correlation (FSC) curves were

4 calculated between the refined model and the half map 1, and between the refined model

5 and the half map 2 (Figs. S3, S5, S6, and S8) (35). The statistics of the model refinement

6 and validation are summarized in Table S1. Figures of the density maps and molecular

7 graphics were prepared using CueMol (http://www.cuemol.org) and UCSF Chimera (36).

9 Bioluminescent ATP release assay

10 The extracellular ATP concentration was measured by the luciferin-luciferase

11 reaction as previously reported (21). HeLa cells seeded at a density of 1.2 × 104 cells

12 well−1 on 96-well cell culture plates (Corning Costar, Corning, NY, USA) were

13 transfected with 0.6 µg pIRES2.AcGFP1 encoding WT or mutant OlCALHM1 cDNA or

14 the empty vector using Lipofectamine 3000 (Thermo Fisher Scientific), according to the

15 manufacturer’s instruction, and incubated for 4 h at 37 ºC. Subsequently, transfection

16 media were replaced with fresh culture media, and cells were incubated at 27 ºC for 4 h

17 before being tested. In experiments shown in Fig. S2A–C, cells were transfected with 0.2

18 µg pIRES2.AcGFP1 encoding HsCALHM1, HsCALHM2, OlCALHM1, or CeCLHM-1

19 cDNA or the empty vector, and incubated in the transfection media for 4 h at 37 ºC and

20 then in fresh culture media for 20 h at 27 ºC. Cells were then washed twice with the

21 standard bath solution and incubated in 100 µL of the standard bath solution for another

22 1 h at room temperature. Immediately after 75 µl of the bath solution was replaced with

2+ 2+ 23 an equal volume of the Ca -free bath solution to make final [Ca ]o ~17 nM, 10 µl of the

24 luciferin-luciferase solution (FL-AAM and FL-AAB, Sigma, St. Louis, MO, USA) was

1 added to each well and the plate was placed in a microplate luminometer (Centro LB960,

2 Berthold Technologies, Bad Wildbad, Germany). Luminescence was measured every 2

3 min at 25 ºC. The extracellular ATP concentration was calculated from a standard curve

4 created in each plate. The standard bath solution contained (in mM): 150 NaCl, 5 KCl, 2

2+ 5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH. The Ca -free

6 bath solution contained (in mM): 150 NaCl, 5 KCl, 5 EGTA, 1 MgCl2, 10 Hepes, and 10

7 glucose, pH 7.4 adjusted with NaOH.

8 9 Preparation of proteoliposomes 10 Lipids from chloroform stocks, mixed at a 2:1 weight ratio of 1-palmitoyl-2-

11 oleoyl-sn-glycero-3-phosphoglycerol (Avanti) with 12% egg phosphatidylcholine:1-

12 palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (Avanti), were dried and solubilized in

13 a solution containing 100 mM KCl, 0.1 mM EGTA, 2.3% n-octyl-β-D-glucopyranoside,

14 and 25 mM HEPES, pH 7.6. Solubilized OlCALHM1EM was added to the lipid-detergent

15 mixture at a 50:1 lipid to protein ratio. After a short incubation, Bio-Beads SM-2 (Bio-

16 Rad) were added and mixed at 4°C overnight. To confirm the reconstitution, after

17 ultracentrifugation (125,000g, 15 min, 4°C) and solubilization in buffer (50 mM Tris–HCl,

18 pH 8.0, 150 mM NaCl, 1.0% DDM, and 0.2% CHS) at 4°C for 1 h, the proteoliposomal

19 solutions were subjected to SDS-PAGE.

20 21 ATP transport assay 22 Liposomes and proteoliposomes were loaded with 1 mM ATP and sonicated for

23 30 s, using a Bioruptor (CosmoBio). After removal of the extraliposomal ATP by

24 chromatography on Sephadex G-50 fine resin (GE Healthcare), 50 μl portions of the

25 samples were combined with 50 μl of a mixture containing 45 nM luciferase (QuantiLum

1 Photinus pyralis recombinant luciferase; Promega, Madison, WI), 1.2 nM luciferin

2 (Nacalai Tesque), 1.0 mM EDTA, and 10 mM MgSO4. After 1h incubation with or

3 without 0.4% Triton X-100, the luminescence was measured on an ARVO-X3 microplate

4 reader (PerkinElmer). The amount of ATP retained in the liposomes was determined as

5 the difference in the luminescence between total ATP and extraliposomal ATP that was

6 accessible to luciferase without Triton X-100.

9 Figure legends:

10 Fig. S1: Amino acid sequence alignment of the CALHM1 orthologs.

11 (A) Alignment of the amino-acid sequences of killifish CALHM1 (OlCALHM1, UniProt

12 ID: H2MCM1), human CALHM1 (UniProt ID: Q8IU99), rat CALHM1 (UniProt ID:

13 D4AE44), cat CALHM1 (UniProt ID: M3VYF2), chicken CALHM1 (UniProt ID:

14 A0A1D5NWS1), and Danio rerio CALHM1 (UniProt ID: E7F2J4). Secondary structure

15 elements for α-helices are indicated by cylinders. Conservation of the residues is indicated

16 as follows: red panels for completely conserved; red letters for partly conserved; and

17 black letters for not conserved. (B and C) Conservation of the residues of CALHM1

18 family members. The sequence conservation among 384 CALHM1 orthologs was

19 calculated using the ConSurf server (http://consurf.tau.ac.il), and is colored from cyan

20 (low) to maroon (high). The residues constituting the subunit interface are indicated by

21 stick models. They are conserved in the CALHM1 orthologs, except for Met187 and

22 Phe194, which are exposed to the membrane environment.

1 Fig. S2: Functional analyses of the CALHM proteins.

2 (A and B) Time courses of extracellular ATP levels due to release from HeLa cells

3 transfected with the empty vector (mock), OlCALHM1, HsCALHM1, HsCALHM2, or

2+ 4 CeCLHM-1 following exposure to essentially zero [Ca ]o (17 nM), in the absence (A) or

5 presence (B) of RUR (20 μM). Data are displayed as means ± S.E.M. (standard error of

6 the mean) (n = 8). (C) Time courses of extracellular ATP levels due to release from the

7 empty vector (mock), full-length OlCALHM1, or OLCALHM1EM transfected HeLa cells,

2+ 8 following exposure to essentially zero [Ca ]o (17 nM). Data are displayed as

9 means ± S.E.M. (n = 8). (D) Permeability of the purified OlCALHM1EM protein to ATP.

10 Liposomes and OlCALHM1EM proteoliposomes loaded with 1 mM ATP were

11 fractionated on the size exclusion column to separate the free extraliposomal ATP from

12 the ATP retained inside. The retained ATP was measured by luminescence using a

13 luciferin/luciferase assay, as the difference between total ATP (inside plus outside,

14 measured after the addition of Triton X-100 to 0.4%) and extraliposomal ATP (before

15 Triton X-100). Data are displayed as the means ± S.E.M. (n = 3) of the ATP retained/total

16 counts.

18 Fig. S3: Cryo-EM analysis of OlCALHM1.

19 (A) Representative cryo-EM image of OlCALHM1, recorded on a 300 kV Titan Krios

20 electron microscope equipped with a K3 camera. (B) Image processing workflow of the

21 single particle analysis. (C) Angular distribution. (D) Fourier shell correlation curve of

22 two half-maps. (E) Map-to-model correlation curves.

1 Fig. S4: Atomic model of OlCALHM1 in the density maps.

2 (A) Cryo-EM density and atomic model of each segment of OlCALHM1EM. All maps are

3 contoured at 4.0 σ. (B) Structural comparison of OlCALHM1EM and Gap-like channels

4 (connexin 26, orange; innexin, light green; and LRRC8, purple).

6 Fig. S5: Cryo-EM analysis of HsCALHM2.

7 (A) Representative cryo-EM image of HsCALHM2, recorded on a 300 kV Titan Krios

8 electron microscope equipped with a Falcon III camera. (B) Image processing workflow

9 of the single particle analysis. (C) Angular distribution. (D) Fourier shell correlation curve

10 of two half-maps. (E) Local resolution analysis. (F) Map-to-model correlation curves.

12 Fig. S6: Cryo-EM analysis of CeCLHM-1.

13 (A) Representative cryo-EM image of CeCLHM-1, recorded on a 300 kV Titan Krios

14 electron microscope equipped with a Falcon III camera. (B) Image processing workflow

15 of the single particle analysis. (C) Angular distribution. (D) Fourier shell correlation curve

16 of two half-maps. (E) Local resolution analysis. (F) Map-to-model correlation curves.

18 Fig. S7: Amino acid sequence alignment of OlCALHM1, HsCALHM2,

19 and CeCLHM-1.

20 Alignment of the amino-acid sequences of killifish CALHM1 (OlCALHM1, UniProt ID:

21 H2MCM1), human CALHM1 (UniProt ID: Q8IU99), human CALHM2 (UniProt ID:

22 Q9HA72), and CeCLHM-1 (UniProt ID: Q18593). Secondary structure elements for α-

23 helices are indicated by cylinders. Conservation of the residues is indicated as follows:

1 red panels for completely conserved; red letters for partly conserved; and black letters for

2 not conserved.

4 Fig. S8: Cryo-EM analysis of the CALHM chimera.

5 (A) Representative cryo-EM image of the CALHM chimera (OlCALHM1c), recorded on

6 a 300 kV Titan Krios electron microscope equipped with a K3 camera. (B) Image

7 processing workflow of the single particle analysis. (C) Angular distributions of the

8 OlCALHM1c 8-mer (left) and 9-mer (right). (D) Fourier shell correlation curves of two

9 half-maps of the OlCALHM1c 8-mer (left) and 9-mer (right). (E) Local resolution

10 analysis of the OlCALHM1c 8-mer (left) and 9-mer (right). (F) Map-to-model correlation

11 curves of the OlCALHM1c 8-mer (left) and 9-mer (right).

13 Fig. S9: Amino acid sequence alignment of OlCALHM1, HsCALHM1,

14 and HsCALHM3.

15 Alignment of the amino-acid sequences of killifish CALHM1 (OlCALHM1, UniProt ID:

16 H2MCM1), human CALHM1 (UniProt ID: Q8IU99), and human CALHM3 (UniProt ID:

17 Q86XJ0). Secondary structure elements for α-helices are indicated by cylinders.

18 Conservation of the residues is indicated as follows: red panels for completely conserved;

19 red letters for partly conserved; and black letters for not conserved.

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Table S1: Cryo-EM data collection, processing, model refinement, and validation statistics. OlCALHM1 HsCALHM2 CeCLHM-1 Chimeric Chimeric construct (8-mer) construct (9-mer) EMDB ID XXXX XXXX XXXX XXXX XXXX PDB ID XXXX XXXX XXXX XXXX XXXX Data collection and processing Microscope Titan Krios G3i Titan Krios G3i Titan Krios G3i Titan Krios G3i Detector Gatan K3 camera FEI Falcon III FEI Falcon III Gatan K3 camera Nominal magnification 105,000 96,000 96,000 105,000 Voltage (kV) 300 300 300 300 Electron exposure (eÅ2) 59 60 50 50 Nominal defocus range (μm) 0.8 to 1.6 0.75 to 1.75 0.75 to 1.75 0.8 to 1.52 Pixel size (Å pixel) 0.83 0.8346 0.8346 0.83 Symmetry imposed C8 C11 C9 C8 C9 Number of movies 3,075 2,110 2,906 2,808 Initial particle numbers 1,231,992 946,353 832,750 1,689,949 Final particle numbers 102,109 365,116 65,887 233,566 227,781 Map resolution (Å) 2.66 3.51 3.60 3.4 3.4 FSC threshold 0.143 0.143 0.143 0.143 0.143 Map sharpening B factor (Å2) 61.6548 203.38 120.37 181.13 184.76 Model building and refinement Model composition

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Protein atoms 17,412 23,716 17,717 11,624 17,334 Other atoms 139 0 0 0 0 R.M.S. deviations from ideal Bond lengths (Å) 0.009 0.004 0.008 0.007 0.008 Bond angles (°) 1.013 0.898 0.831 0.879 0.924 Validation Clashscore 4.85 3.00 2.57 2.21 5.28 Rotamer outliers (%) 2.23 0.00 0.94 0.63 0.49 Ramachandran plot Favored (%) 97.3 91.55 94.24 98.35 97.48 Allowed (%) 2.7 8.45 5.76 1.65 2.52 Outlier (%) 0.0 0.00 0.00 0.00 0.00

A B Extracellular C region

2.4 (15 Å) OUT

2.7 Membrane region 3.0 (37 Å) 90° 3.3

IN 3.6 Resolution (Å) Cytoplasmic region (28 Å) 110 Å

D E

Extracellular region EL2H EL2H OUT OUT

G TM3c H

N TM3c TM1 TM2a Membrane region TM3b TM4 TM1 TM2a N 90° NTH TM4 TM3b NTH

TM3a TM3a

IN TM2b IN Cytoplasmic region TM2b CTH F C CTH

F R105 TM4 H G EL2H R203 K122 TM2a N71 C205 C100 K178 Y97 C160 C43 N73 N72 M98 T207 T214 F211 G121 S218 C41 V94 TM4 D120 R225 V76 K213 I221 TM1 W217 C126 K90 TM3b Y34 TM2a T85 TM3c

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A C L212 F D H219 D229 D K213 F228 C232 E254 Y247 I224 Y220 F228 T231

E F248 H235 C243 B

B TM2 C E F Outside lipid Y176 Y47 N131 F128 I180 T50 R175 F124 TM4 TM3 Y49 C179 C41 Q182 Y51 K178 CHS1 CHS2 E38 S36 V116 M119 L55 CHS3 L56 W186 F37 P59 Q182 L189 M187 L58 V114 M33 TM1 L189 L28 TM1 L190 I61 W62 T193 TM1 M192 F194 L107 L66 F197 CHS4 Q12 R200 F68 R200 R200 V69 M19 TM3 N72 S13 TM4 Q104 TM4

Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A

OUT

15.7 Å Q9 Q12 S13

B C Radius (Å)

Gln9 ΔN11

Distance (Å) Gln12 Ser13

ΔN19

D Ca2+ removal E No treatment (Ca2+ bound)

Mock Mock OlCALHM1 WT OlCALHM1 WT 100 OlCALHM1 ΔN11 100 OlCALHM1 ΔN11 OlCALHM1 ΔN19 OlCALHM1 ΔN19 80 80

60 60

40 40 [ATP]o (nM) [ATP]o (nM) 20 20

0 0 0 20 40 60 0 20 40 60 time (min) time (min)

Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this A preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowedC without permission. OlCALHM1 HsCALHM2 CeCLHM-1 OlCALHM1 HsCALHM2 CeCLHM-1

Out D

16 Å 64 Å 44 Å 2 3 4 1 NTH 1 4 3 1 4 In 2 4 2 3 2 3 CTH

180°

90°

3 4 34 Å 2 60 Å 90 Å 68 Å

CTH 1

110 Å 143 Å 126 Å B D

OlCALHM1 HsCALHM2 CeCLHM-1

TM3 Out TM4’ TM4 TM2 W186 W189 4’ 2 3 4 W113 4’ 2 3 4 W195 4’ 2 3 4 W117 1 P59 W118 NTH

1 93 Å 93 Å P64 P64 N71 P110 In P114 CTH C211 N76 CTH P115 CTH L205 16.8 Å N76 17.9 Å I202 14.4 Å

E OlCALHM1 HsCALHM2 CeCLHM-1

Y185 Y176 R179 R52 Y47 L52 C179 Y182 E189 A188 Y51 Y56 H56 T50 E183 L55 Y55 I180 I192 F60 Q191 Q182 L186 Q185 A183 L55 A59 A60 V59 G54 G196 W195 W189 I58 W186 L190 V63 W118 M187 W113 W117 L199 G63 L198 P59 L189 P64 I192 P64 L190 G193 A66 I61 W62 L66 V67 V203 A67 G202 T193 I197 A196 L70 F194 L65 P110 P110 L66 I70 P114 P114 I71 P115 P115 I71 A205 A196 V199 F206 F197 180° V69 F200 180° I73 180° T74 F68 I73 I74

TM4’ TM2 TM3 TM3 TM2 TM4’ TM4’ TM2 TM3 TM3 TM2 TM4’ TM4’ TM2 TM3 TM3 TM2 TM4’

F OlCALHM1 HsCALHM2 CeCLHM-1 E254 W278 F258 K213 Y154 Y296 W302 H300 W267 Y219 R215 F256 M255 Y223 W262 Y216 F248 Y225 V270 I244 E227 D228 Y229 Y220 F251 F231 H235 F228 A243 H238 F257 F237 N226 E233 A249 A240 H219 D229 H244 V240 E224 Q229 R240 E250 K236 180° 180° 180° 90° 90° 90°

W217 W220 D229 H219 D228 Y219 V221 R215 Q229 Y225 E224 N226 F228 Y220 Y229 A235 E233 E227 F231 K236 I244 Y216 R240 H300 F237 T231 A240 F256 F248 F250 H238 L242 L248 N252 F239 F251 F257 L248 E250 H235 H244 Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this A preprint (which was not certified by peer review) is the author/funder. All rights reserved. NoC reuse allowedOlCALHM1c without 8-mer permission. OlCALHM1c 9-mer 8-mer 9-mer

Out D

1 441 2 3

In CTH

B D

Y176 Y47 Out C179 I180 T50 A183 G54

60 Å 60 Å M187 I58 4’ 1 4 1 2 4’ 2 4 91 Å L190 I61 P110 3 3 F194 L65 In CTH F197 I202 F68 0.6 Å TM3’ TM4’ TM2 TM3 TM4 114 Å 119 Å

90° E

5 6 4 4 5 34 Å 42 Å 180° 3 3 6 7 68 Å 2 7 2 1 8 8 1 9 TM4’ TM1 TM4

TM2 TM3

Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.31.928093; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A TM3c OPEN TM1

TM3b

NTH

B TM3c

CLOSE TM1

TM3b

NTH

Figure 6