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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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 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 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. 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 3 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 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. 8 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 4 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. 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. 12 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 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. 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.
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