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Three-Dimensional Structure of a Human Connexin26 Gap Junction Channel Reveals a Plug in the Vestibule

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Three-Dimensional Structure of a Human Connexin26 Gap Junction Channel Reveals a Plug in the Vestibule Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule Atsunori Oshima†‡, Kazutoshi Tani†, Yoko Hiroaki†‡, Yoshinori Fujiyoshi†‡§¶, and Gina E. Sosinsky¶ʈ †Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; ʈNational Center for Microscopy and Imaging Research, Department of Neurosciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0608; ‡Core Research for Evolution Science and Technology (CREST), Japan Science and Technology Agency (JST), Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; and §Japan Biological Information Research Center (JBIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6, Aomi, Koto-ku, Tokyo 135-0064, Japan Communicated by David J. DeRosier, Brandeis University, Waltham, MA, April 24, 2007 (received for review January 29, 2007) Connexin molecules form intercellular membrane channels facili- that the relatively unordered carboxyl terminus of larger iso- tating electronic coupling and the passage of small molecules forms interferes with the necessary tight packing in a crystal. In between adjoining cells. Connexin26 (Cx26) is the second smallest addition, we used a site-specific mutant of human Met-34, member of the gap junction protein family, and mutations in Cx26 hCx26M34A, because this mutant expresses in baculovirus in- cause certain hereditary human diseases such as skin disorders and fected Sf9 cells at higher quantities than wild-type Cx26-infected hearing loss. Here, we report the electron crystallographic struc- cells. The hCx26M34A mutant is a single-site mutation at the ture of a human Cx26 mutant (M34A). Although crystallization same position as the hCx26M34T mutant, which can cause trials used hemichannel preparations, the density map revealed prelingual nonsyndromic hereditary deafness (11). Although not that two hemichannels redocked at their extracellular surfaces into as well characterized as hCx26M34T (12), this single-point full intercellular channels. These orthorhombic crystals contained mutation of a Met to Ala decreases dye coupling in exogenously two sets of symmetry-related intercellular channels within three transfected HeLa cells and forms structures indistinguishable lipid bilayers. The 3D map shows a prominent density in the pore from wild-type gap junctions (13). We succeeded in making 2D of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. crystals of the hCx26M34A suitable for cryoelectron crystallog- The density map suggests that physical blocking may play an raphy. The 3D structure of hCx26M34A gap junction channels important role that underlies gap junction channel regulation. Our reveals a prominent density in the pore of each hemichannel, structure allows us to suggest that the two docked hemichannels suggesting that the channel is blocked by a physical obstruction. can be independent and may regulate their activity autonomously with a plug in the vestibule. Results Two-Dimensional Crystallization of Connexin26 Complexes. connexin channels ͉ electron crystallography ͉ intercellular hCx26M34A gap junction channels were expressed in and iso- communication ͉ membrane protein structure ͉ two-dimensional crystals lated from Sf9 insect cells [see supporting information (SI) Fig. 7A]. Hemichannels (connexons) were isolated by affinity puri- ap junctions contain intercellular communication channels fication using a C-terminal hexa-histidine tag (SI Fig. 7B). Gthat allow a wide variety of solutes with different sizes to be Purified hemichannels were mixed with the lipid dioleoylphos- transferred between the cytoplasm of adjacent cells. These phatidylcholine (DOPC) at a lipid-to-protein ratio of 1 (wt/wt). solutes include ions, metabolites, nucleotides, peptides, and Reconstitution into lipid bilayers by dialysis produced 2D crys- secondary messengers. Gap junction channels have critical roles tals Ͼ1 ␮m in diameter (SI Fig. 8A). Although the purified in many biologically important processes including cardiac de- hemichannel is hexameric, the 2D arrays obtained by dialysis velopment, fertility, the immune system, and electrical signaling showed an orthorhombic crystal lattice (SI Fig. 8B). in the nervous system (1). The diversely expressed connexin26 We recorded images of the hCx26M34A crystals embedded in (Cx26) is the second-smallest member of the conserved mam- 0.05–1% tannic acid, 2–40% trehalose, or the combination of malian gap junction protein family. Hereditary mutations in them. Computed diffraction patterns of one of the best 0° images human Cx26 cause macroscopic symptoms such as certain skin showed reflections to a resolution of Ϸ11Å(SI Fig. 8C). After disorders and nonsyndromic and syndromic deafness (2). Early electron microscopic studies suggested that closure of image processing to correct crystal distortions, the resolution the gap junction channel occurs by rotating all six subunits in improved to 7 Å (SI Fig. 8D). Image processing revealed that the ϭ each hemichannel (3, 4). More recently, a 3D structure of a crystals had p22121 symmetry and unit cell parameters of a ϭ ␥ ϭ truncated form of Cx43 reported on the arrangement and the 112.4 Å, b 111.2 Å, and 90°. assignments of the four transmembrane ␣-helical bundle in each connexin (5, 6). Electrophysiological experiments have shown Author contributions: A.O. and Y.F. designed research; A.O., K.T., Y.H., Y.F., and G.E.S. that the voltage sensor involves charged residues in the connexin performed research; A.O., K.T., Y.H., Y.F., and G.E.S. analyzed data; and A.O., Y.F., and N terminus and that it appears to face the aqueous pore (7, 8), G.E.S. wrote the paper. and studies of hemichannel conductivity favor ‘‘an individual The authors declare no conflict of interest. subunit gating model’’ (9). In addition, the selectivity of gap Freely available online through the PNAS open access option. junctions for permeation of small molecules under Ϸ1 kDa (also Abbreviations: Cx: connexin, Cx26M34A: connexin26 site-specific mutant Met34Ala. known as ‘‘permselectivity’’) depends on the connexin isoform. Data deposition: The cryoEM structure reported in this paper has been deposited in the Each isoform has unique physiological responses to ions, phos- Macromolecular Structure Database (MSD), www.ebi.ac.uk/msd-srv/emsearch/index.html phorylation, and pH. Permselectivity is hypothesized to occur (accession no. EMD-1341). independently from voltage gating, implying that gap junction ¶To whom correspondence may be addressed. E-mail: [email protected] or yoshi@ channels possess multiple gating mechanisms (10). em.biophys.kyoto-u.ac.jp. In this study, we focus on the structure of Cx26 gap junction This article contains supporting information online at www.pnas.org/cgi/content/full/ channels, because the short cytoplasmic tail of Cx26 makes it 0703704104/DC1. more amenable for the formation of 2D crystals. It is believed © 2007 by The National Academy of Sciences of the USA 10034–10039 ͉ PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703704104 Downloaded by guest on September 28, 2021 BIOPHYSICS Fig. 1. Three-dimensional structure of Cx26 orthorhombic crystals.(A) Molecular packing of Cx26 in the 2D crystal. The gap junction channels are incorporated in three lipid bilayers (Mem-1–Mem-3) with 21 symmetry along Mem-2. (B) View of the Cx26 density map perpendicular to the membrane plane. The three membranes, indicated by gray bars, surround two extracellular gap regions. The map is contoured at 1.0␴ (light blue) and 2.4␴ (wheat color) above the mean density. The transmembrane ␣-helical ribbon model (6) is docked into the density for one of the hemichannels. The four helices are color-coded as in Fig. 2. Two helices D make contact with adjacent gap junction channels (red arrows). (Scale bar, 40 Å.) (C and D) Forty-angstrom-thick sections through the density map parallel to the membrane plane, showing protein embedded in membranes Mem-1 (identical to Mem-3) (C) and Mem-2 (D). Tail ends of two helices D are indicated by red arrows as in B. (Scale bars, 40 Å.) Three-Dimensional Structure Determination and Organization of that the crystals have a thickness of Ϸ240 Å and contain three hCx26M34A Orthorhombic Crystals. To determine a 3D structure, lipid bilayers (labeled Mem-1, Mem-2, and Mem-3 in Fig. 1). we collected images of samples tilted up to 45° and combined Remarkably, the map also shows that the hemichannels re- them to produce a density map at a resolution of 10 Å in the docked through their extracellular surfaces, forming complete membrane plane and 14.1 Å normal to the membrane plane (Fig. gap junction channels (Fig. 1 A and B). This is consistent with 1, SI Table 1, and SI Fig. 8E). A side view of the 3D map reveals published results proposing extensive hydrophobic surfaces in Oshima et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 10035 Downloaded by guest on September 28, 2021 Fig. 2. Structural details of the Cx26 gap junction. The map is contoured as in Fig. 1. (Inset) Twenty-angstrom-thick section perpendicular to the membrane plane through the density map of a hemichannel in Mem-2. This section corresponds to the region enclosed by the white lines shown in A. The arrowhead points to the large density in the pore. The inner cytoplasmic protrusions (white arrows) extend from the cytoplasmic ends of helices B and C. (A–C) Thirty-angstrom-thick slabs through the density map corresponding to the position of the lines shown in Inset. The four helices are labeled A (cyan, AЈ), B (green, BЈ), C (yellow), and D (pink) as in the original Cx43 structure (5). The arrowhead and white arrows represent the plug and the inner cytoplasmic protrusions, respectively,asinInset. the gap region (14). In bilayers Mem-1 and Mem-3, the ture made it possible to unambiguously dock the proposed hemichannels show poorer density than in Mem-2 (Fig.
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