cryo-em structures of calcium homeostasis modulator ... · 2 calcium homeostasis modulator (calhm)...

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Title: 1 Cryo-EM structures of calcium homeostasis modulator channels 2 in diverse oligomeric assemblies 3 4 One Sentence Summary: 5

Cryo-EM structures reveal the ATP conduction and oligomeric assembly 6 mechanisms of CALHM channels. 7

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

preprint (which was not certified by peer review) is the author/funder.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

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Abstract: 1

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

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

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

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

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

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

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

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

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

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

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

words). 13

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Main Text: 1

Introduction 2

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

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

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

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

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

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

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

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

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

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

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

concentration ([Ca2+]o) reduction increase the open probability of CALHM channels (8), 14

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

channels. 7

8

Structure determination of CALHM1 9

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

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

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

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

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

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

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

the luciferin–luciferase reaction (5) (Fig. S2A–C). Lowering [Ca2+]o induced a larger 17

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

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

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

channel activated by low [Ca2+]o, as also reported for HsCALHM1 (5). 21

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

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

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


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ATP-release activity elicited by low [Ca2+]o when expressed in HeLa cells (Fig. S2C). 1

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

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

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

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

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

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

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

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

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

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

12

Structure of OlCALHM1 13

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

probably have an important role in channel function. 7

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

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

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

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

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

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

structural organization of the TM helices inside the channel. 14

15

Subunit interface in OlCALHM1 16

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

stabilization of the CALHM1 channels. 17

18

Pore architecture of OlCALHM1 19

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

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

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

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

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


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

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

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

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

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

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

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

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

agreement with the previous functional analysis of HsCALHM1. 9

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

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

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

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

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

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

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

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

associated with the Ca2+-dependent channel closing. 18

19

Structures of HsCALHM2 and CeCLHM-1 and structural differences 20

among CALHM homologs 21

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

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

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


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

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

extracellular ATP concentration in response to lowering [Ca2+]o compared to mock-3

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

channels activated by low [Ca2+]o, as in the CALHM1 channels (Fig. S2A, B). To further 5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

family members. 14

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

11

Oligomeric assembly mechanism of CALHM channels 12

The three different oligomeric assembly structures of the CALHM homologs 13

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(14). 24


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1

Discussion 2

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Given the structural and sequence similarities between OlCALHM1 and HsCALHM2 24


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

proteins. 16

17

18

References and Notes: 19

1. G. Burnstock, Historical review: ATP as a neurotransmitter. Trends in 20 Pharmacological Sciences. 27, 166–176 (2006). 21

2. M. P. Abbracchio, G. Burnstock, A. Verkhratsky, H. Zimmermann, Purinergic 22 signalling in the nervous system: an overview. Trends in Neurosciences. 32, 19–23 29 (2009). 24

3. A. Taruno, ATP release channels. International Journal of Molecular Sciences. 25 19, 808 (2018). 26

4. U. Dreses-Werringloer, J. C. Lambert, V. Vingtdeux, H. Zhao, H. Vais, A. 27


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10

11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

no financial or other interest in this research. 4

Data and materials availability: 5

6

Figure legends: 7

Fig. 1: Overall structure of OlCALHM1. 8

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

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

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

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

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

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

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

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

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

TMs around Asp120 (H). 18

19

Fig. 2: Subunit interfaces between two adjacent subunits. 20

(A) Overview between two adjacent subunits, viewed from the extracellular side. (B–D) 21

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

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

CHS molecules are indicated by grey stick models. 24


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1

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

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

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

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

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

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

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

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

(D) or normal (1.9 mM) [Ca2+]o (E). Data are displayed as means ± S.E.M. n = 15 (Mock), 10

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

12

Fig. 4: Structural differences among OlCALHM1, HsCALHM2, and 13 CeCLHM-1. 14

(A) Structural comparisons among OlCALH1, HsCALHM2, and CeCLHM1. Overall 15

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

9

Fig. 5: Structures and oligomerization states of the chimeric CALHM 10 proteins. 11

(A) EM density maps of the OlCALHM1c 8-mer (left) and 9-mer (right), colored 12

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

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

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

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

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

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

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

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

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

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

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

24


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Fig. 6: Insight into channel gating 1

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

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

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

state, TM1 blocks the pore (B). 5


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Supplementary Materials: 1

2

Materials and Methods: 3

Expression and purification of OlCALHM1, HsCALHM2, and the 4

chimeric construct OlCALHM1c 5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

7

Expression and purification of CeCLHM-1 8

The Caenorhabditis elegans calcium homeostasis modulator protein (CeCLHM-9

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

pooled. 10

11

Grid preparation 12

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

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

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

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

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

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

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

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

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

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

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

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


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

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

100% humidity conditions. 3

4

EM image acquisition and data processing 5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

by CTFFIND 4.1 (28). 23

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

workflow is described in Fig. S5. 20

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


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

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


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

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

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

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

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

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


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

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

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

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

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

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

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

workflow is described in Fig. S8. 15

16

Model building and refinement 17

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

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

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

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

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

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

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


https://doi.org/10.1101/2020.01.31.928093

30

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

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

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

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

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

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

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

8

Bioluminescent ATP release assay 9

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

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

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

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

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

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

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

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

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

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

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

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

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

an equal volume of the Ca2+-free bath solution to make final [Ca2+]o ~17 nM, 10 µl of the 23

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


https://doi.org/10.1101/2020.01.31.928093

31

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

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

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

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

CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH. The Ca2+-free 5

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

glucose, pH 7.4 adjusted with NaOH. 7

8

Preparation of proteoliposomes 9

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

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

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

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

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

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

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

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

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

solutions were subjected to SDS-PAGE. 19

20

ATP transport assay 21

Liposomes and proteoliposomes were loaded with 1 mM ATP and sonicated for 22

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

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

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


https://doi.org/10.1101/2020.01.31.928093

32

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

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

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

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

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

accessible to luciferase without Triton X-100. 6

7

8

Figure legends: 9

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

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

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

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

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

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

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

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

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

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

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

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

Phe194, which are exposed to the membrane environment. 22

23


https://doi.org/10.1101/2020.01.31.928093

33

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

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

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

CeCLHM-1 following exposure to essentially zero [Ca2+]o (17 nM), in the absence (A) or 4

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

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

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

following exposure to essentially zero [Ca2+]o (17 nM). Data are displayed as 8

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

Liposomes and OlCALHM1EM proteoliposomes loaded with 1 mM ATP were 10

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

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

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

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

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

counts. 16

17

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

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

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

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

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

23


https://doi.org/10.1101/2020.01.31.928093

34

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

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

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

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

5

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

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

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

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

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

11

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

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

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

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

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

17

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

and CeCLHM-1. 19

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

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

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

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


https://doi.org/10.1101/2020.01.31.928093

35

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

not conserved. 2

3

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

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

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

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

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

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

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

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

12

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

and HsCALHM3. 14

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

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

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

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

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


https://doi.org/10.1101/2020.01.31.928093

36

Table S1: Cryo-EM data collection, processing, model refinement, and validation statistics. 1

OlCALHM1 HsCALHM2 CeCLHM-1 Chimeric construct (8-mer)

Chimeric 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 (e��Å�2) 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


https://doi.org/10.1101/2020.01.31.928093

37

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

1


https://doi.org/10.1101/2020.01.31.928093

90°

TM

1

TM

2a

TM

3b TM

4

CTH

TM2b

TM3a

N

C

NT

H

TM

3c

Extracellular region

Membrane region

(15 Å)

Cytoplasmic region(28 Å)

(37 Å)

OUT

IN

Extracellular region

Cytoplasmic region

Membrane region

110 Å

N

OUT

IN

OUT

IN

C

90°

C160C160

C41

C126C126

C43

Resolution (Å)

2.4

2.7

3.0

3.3

3.6

N71

R105

Y97

T207

R203

C205C100

M98F211T214

W217

S218

R225I221

K213V94

K90

V76

N72 N73

T85

H

K178

D120D120

K122K122

EL2H

G121G121

Figure 1

TM3b

B

ED

G

C

F

A

NTH

TM1 TM2aTM3b

TM3c

TM4TM4

TM3a

CTHTM2b

EL2HEL2H

G H

Y34

F

TM1TM4TM4

TM2a

TM2a

EL2HEL2H

TM3c

TM4TM4


https://doi.org/10.1101/2020.01.31.928093

Outside lipid

TM4TM4

TM2

Y51Q182Q182

M33

W62

L58

F37M119M119

W186W186

L189L189L190L190

N72R200R200

M187M187

F68

T50

L55

P59

I61

L66

V69

F197F197

T193T193

I180I180

C179C179

F194F194

Y176Y176 Y47B

C

E

Figure 2

D229H219H219

V114

L56

TM3

Y49

TM1

TM1

TM4

L28

CHS2CHS1

R200

TM3

S36

L189

V116

R200

M192

Q104

L107

N131F128

F124 TM4

TM1TM1

CHS3

CHS4Q12

S13

Y220Y220

F228F228

Y247

C243H235H235F248

T231T231

F228

L212L212

I224C232C232

M19

K213

E254

C41

E38

Q182

K178

R175

C F

D

E

D

F

A

B


https://doi.org/10.1101/2020.01.31.928093

Gln9Gln12

Ser13

Radius (Å)

Dis

tanc

e (Å

)

OUT

IN

ΔN11

ΔN19

Figure 3

Q9

Q12S13

A

15.7 Å

B C

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

0 20 40 600

20

40

60

80

100

time (min)

[AT

P]o

(nM

)

Mock

OlCALHM1 WT

OlCALHM1 ΔN11

OlCALHM1 ΔN19

0 20 40 600

20

40

60

80

100

time (min)

[AT

P]o

(nM

)

Mock

OlCALHM1 WT

OlCALHM1 ΔN11

OlCALHM1 ΔN19


https://doi.org/10.1101/2020.01.31.928093

14.4 Å

N71N76N76

17.9 Å

16.8 Å

Y216

Y220 E224

A240

F228

H235F248

Y219 Q229

E227

H300F231

H238F251

Y225Y229

E233

N252

F237

H244F257

Y225Y229

E233 A249

F237

H244

F257

Y219Y223

E227

A243 F231H238F251

Y216

Y220

E224A240

F228H235

F248

H219 D229

E254

K213

F239

W217

T231I244

M255

F258 Y154

I244

H219D229

W278W302

H300Y296

R240

N226

Q229

R215

D228

R215D228W220

L242F250

A235

R240

N226

E250

K236

V240

F256V270

W262

W267

E250

K236

L248F256

V221

W113P59

Y51

P110 L66

TM2 TM3TM4’

P110

L55

W62

V69

Q182

W186

L189

T193

A196

Y47

T50

G54

I58

I61

L65

F68

Y176

I180

C179

A183

M187

L190

F194

F197

TM2 TM3TM4’

P114

R52

L55

A59

V63

L66

I70

I73

R179

E183Y182

L186

L190

G193

I197

F200

W117P64

Y56

P114 I71

A60

V67

I74

Q185

W189

I192

A196

V199

TM2 TM3TM4’

P115

L52

Y55

V59

G63

A66

L70

I73

Y185

E189 A188

I192

G196

L199

V203

F206

W118

P64

H56

P115 I71

F60

A67

T74

Q191

W195

L198

G202

A205

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

2 3 4

CTH

1

2

34

CTH 1

2 3 4

CTH

2 3 4

CTH

2 3 4

CTH

1

1NTH

4’4’

4’

23

41

2 3

41

2 3

4

NTH

I202

C211

TM2

TM3TM4TM4’

W186W189W195

P59P64P64

W113W117W118

P110P114P115

A

B

COlCALHM1 HsCALHM2 CeCLHM-1

E

Figure 4

W113P59

Y51

P110 L66

TM2 TM3TM4’

P110

L55

W62

V69

Q182

W186

L189

T193

A196

Y47

T50

G54

I58

I61

L65

F68

Y176

I180

C179

A183

M187

L190

F194

F197

TM2 TM3TM4’

P114

R52

L55

A59

V63

L66

I70

I73

R179

E183Y182

L186

L190

G193

I197

F200

W117P64

Y56

P114 I71

A60

V67

I74

Q185

W189

I192

A196

V199

TM2 TM3TM4’

P115

L52

Y55

V59

G63

A66

L70

I73

Y185

E189 A188

I192

G196

L199

V203

F206

W118

P64

H56

P115 I71

F60

A67

T74

Q191

W195

L198

G202

A205

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

180° 180° 180°

180°

90° 90° 90°

Out

In

Y216

Y220 E224

A240

F228

H235F248

Y219 Q229

E227

H300F231

H238F251

Y225Y229

E233

N252

F237

H244F257

Y225Y229

E233 A249

F237

H244

F257

Y219Y223

E227

A243 F231H238F251

Y216

Y220

E224A240

F228H235

F248

2 3 4

CTH

2 3 4

CTH

2 3 4

CTH

1

1NTH

23

41

2 3

41

2 3

4

F

D

90°

14.4 ÅI202

L205C211

4’4’

4’

2 3 4

CTH

1

2

34

CTH 1

TM2

TM3TM4TM4’

Out

In

OlCALHM1 HsCALHM2 CeCLHM-1



OlCALHM1HsCALHM2CeCLHM-1

W186W189W195

P59P64P64

W113W117W118

P110P114P115

D

180° 180° 180°

NTH

H219 D229

E254

K213

F239

W217

T231I244

M255

F258 Y154

I244

H219D229

W278W302

H300Y296

R240

N226

Q229

R215

D228

R215D228W220

L242F250

A235

R240

N226

E250

K236

V240

F256V270

W262

W267

E250

K236

L248F256

V221

16 Å

110 Å

64 Å

143 Å

44 Å

126 Å

60 Å 90 Å 68 Å34 Å

93 Å

93 Å

N71N76N76

17.9 Å

16.8 Å


https://doi.org/10.1101/2020.01.31.928093

0.6 Å

2 341

CTH

TM2 TM3

TM4TM1TM4’

I202

TM2 TM3TM4’ TM4

P110

Y47

T50

G54

I58

I61

L65

F68

Y176

I180C179

A183

M187

L190

F194

F197

TM3’

2

3

412

3

41

CTH

4’4’

A

B

Figure 5

C

D

Out

In

TM2 TM3TM4’ TM4

1

2

3

4 5

6

7812

3

45 6

7

89

TM2 TM3

TM4TM1

2

3

412

3

41

CTH

P110

Y47

T50

G54

I58

I61

L65

F68

Y176

I180C179

A183

M187

L190

F194

F197

Out

In

TM3’

I2020.6 Å

4’ 4’

8-mer 9-mer

TM4’

E90°

OlCALHM1c 8-merOlCALHM1c 9-mer

D

180°

2 341

CTH

114 Å 119 Å

34 Å 42 Å

68 Å

60 Å

60 Å

91 Å


https://doi.org/10.1101/2020.01.31.928093

NT

HTM3b

TM

1

TM3c

TM3b TM

1

TM3c

OPEN

CLOSE

NTH

B

A

Figure 6


https://doi.org/10.1101/2020.01.31.928093

cryo-em structures of calcium homeostasis modulator ... · 2 calcium homeostasis modulator (calhm)...

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