Sequence alignment of the <i>Bg</i>AChBP subunits and <i>Ls</i>AChBP (from <i>Lymnaea stagnalis</i>).

<p>The red residues are addressed in the main text in the context of ligand binding (blue boxes), inter-pentamer linkage (red boxes), N-glycan binding (black boxes), or disulfide bridges (arrow symbols). The blue residues probably form salt bridges between adjacent subunits within the same pentamer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g004" target="_blank">Fig. 4E</a>). Note the specific exchanges Y92→F92 in <i>Bg</i>AChBP1 and Y193→F193 in <i>Bg</i>AChBP2. Also note the strictly conserved disulfide bridges stabilizing the eponymous Cys-loop L7 and the gating C-loop L10, the putative additional disulfide bridge C16↔C64 in <i>Bg</i>AChBP1, and the single cysteine C71 in <i>Bg</i>AChBP2. (Chain-specific residue numbers are given.) The secondary structure elements predicted from the published crystal structures are also indicated (L, loop). The short helix following strand β2 and marked in blue is absent in the molecular models of the BgAChBP subunits. Genbank entries JQ814367, JQ814368, AAK64377.</p

Radial phylogenetic tree of AChBP, ACCBP and AChR-LBD.

<p>Sequences of gastropod AChBP and ACCBP polypeptides (marked in red) are compared here to sequences of gastropod AChR-LBD polypeptides (marked in black). Sequences from the pearl oyster <i>P. fucata</i>, the polychaete annelid <i>C. telata</i> and the electric ray <i>T. marmorata</i> are also included (marked in blue). Nodes bootstrap-supported above 900 are indicated by a circle, those above 990 are additionally marked by an asterisk (1000 replicas were calculated). Note that the gastropod AChBP complex is clearly separated from the gastropod nAChR-LBD complex. Also note that <i>Bg</i>AChBP1 and <i>Bg</i>AChBP2 show a clear sister-group relationship, suggesting that they arose from a gene duplication event that occurred within the Planorbidae. The neighbor-joining method implemented in Clustal W was applied. A corresponding identity matrix is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-t002" target="_blank">Table 2</a>. <i>Ac</i>, <i>Aplysia californica</i> (genbank entries AAL37250, AAL37251, AAL78648, AAL78649); <i>Bg</i>, <i>Biomphalaria glabrata</i> (JQ814367, JQ814368); <i>Bt</i>, <i>Bulinus truncatus</i> (PDB-ID 2BJ0); <i>Ct</i>, <i>Capitella teleta</i> (EY637248); <i>Hdd</i>, <i>Haliotis discus discus</i> (ABO26693); <i>Hdh</i>, <i>Haliotis discus hanei</i> (ABU51880, ABU62818); <i>Ls</i>, <i>Lymnaea stagnalis</i> (AAK64377, ABA60380 to ABA60390); <i>Pf</i>, <i>Pinctada fucata</i> (ABF13208); <i>Tm</i>, <i>Torpedo marmorata</i> (PDB-ID 2BG9); LBD, ligand binding domain.</p

Fragments of <i>Bg</i>AChBP1 and <i>Bg</i>AChBP2 as detected in rosette protein material.

<p>With one exception (asterisk), the fragments were obtained by mass spectrometry (nanoUPLC-ESI Q-TOF) after tryptic digestion of a 31 kDa band cut out from an SDS-PAGE gel. The marked sequence was obtained six years earlier by N-terminal protein sequencing of the 31 kDa band. For localization of the peptides, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g002" target="_blank">Fig 2</a>.</p

Homology models of <i>Bg</i>AChBP1.

<p><b>(A)</b> The modeled subunit showing the N-terminal helix α1, the 10-stranded β-sandwich, the connecting loops L1 to L10, the three disulfide bridges, and the potential attachment site for N-linked glycans. <b>(B–D)</b> The modeled pentamer in side view (B) and the two different top views (C, D). The C-face is defined by the five C-termini and eponymous Cys-loops L7, the N-face contains the five N-termini and α1 helices. <b>(E)</b> Two neighboring subunits extracted from the modeled pentamer, with amino acid residues in the principal side of the ligand-binding pocket highlighted. Note that instead of phenylalanine F92, other AChP-LBD/AChBP members possess a tyrosine. Putative salt bridges connecting both subunits are also shown. <b>(F)</b> The modeled subunit showing the three disulfide bridges and the amino acids presumably involved in inter-pentamer contacts. Red labels mark features that are specific for <i>Bg</i>AChBP. (PDB-ID of the BbAChBP1 pentamer: 4AOD; PDB-ID of the <i>Bg</i>AChBP2 pentamer: 4AOE).</p

Identity matrix of AChBP, ACCBP and nACHR-LBD proteins.

<p>Values were calculated using the CLUSTAL W multiple sequence alignment underlying the phylogenetic tree shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g012" target="_blank">Fig. 12</a>.</p

Gene structure of <i>Bg</i>AChBP1 and <i>Bg</i>AChBP2.

<p>Data retrieved from the preliminary <i>B. glabrata</i> genomic data (<a href="http://129.24.144.93/blast_bg/2index.html" target="_blank">http://129.24.144.93/blast_bg/2index.html</a>). Exon 1 and the first three amino acids encoded by exon 2 belong to the signal peptide, as deduced from evaluation in SignalP, and N-terminal protein sequencing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-t001" target="_blank">Table 1</a>). Genbank entries JQ814367, JQ814368.</p

3D-EM processing of BgAChBP.

<p>Characteristic class sum images (top), and the corresponding reprojections (bottom) of the density map shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g008" target="_blank">Fig. 8A–D</a>. Note peripheral protrusions in class sum images (upper arrow) that are absent in the corresponding reprojections (lower arrow). This disappearance results from masking for avoiding noise bias (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#s4" target="_blank">Methods</a>).</p

Electron microscopy of recombinant <i>Bg</i>AChBP1 and BgAChBP2 as expressed in <i>E. coli</i>.

<p><b>(A)</b> Recombinant BgAChBP1 pentamers (short arrow) and dodecahedra (large arrows). Left insert, enlarged view along the five-fold symmetry axis of a recombinant dodecahedron and a single pentamer, respectively. Right insert, 3D reconstruction (resolution ∼20 Å) from ∼3000 negatively stained particles of the recombinant <i>Bg</i>AChBP1 dodecahedron. <b>(B)</b> Recombinant BgAChBP2 pentamers (short arrow) and presumed di-pentamers (large arrows). In several independent expression experiments, not a single dodecahedron was detected in the electron microscope.</p

Putative inter-pentamer interfaces in a <i>Bg</i>AChBP1 dodecahedron.

<p><b>(A)</b> Molecular model of the dodecahedron, viewed along one of the three-fold symmetry axes. The amino acid appositions providing opportunities for inter-pentamer bonding are highlighted. <b>(B)</b> The same view as in (A), with most of the three pentamers joined in the vertex removed. They are indicated by the yellow, light blue and orange red helices α1. In the center, the trigonal ring that makes the inter-pentamer contact is visible. <b>(C)</b> The residues that together with helix α1 form the trigonal ring, shown in ball & stick mode. In addition, loops L1 and L3 are indicated. Subunits of similar color stem from the same pentamer. Note the position of the C16↔C64 bridge (arrows). <b>(D)</b> Details of the central and three adjacent trigonal rings. Each ring connects three pentamers at their common vertex (<i>via</i> the F71 cluster, three salt bridges R3↔E70 and three salt bridges D25↔R63). Alternatively, two parallel salt bridges D25↔R63 can be considered as connection between two adjacent pentamers across their common edge. (PDB-ID of the BbAChBP1 pentamer: 4AOD).</p

Cryo-EM structures of the <i>Bg</i>AChBP dodecahedron.

<p><b>(A)</b> The final 6-Å cryo-EM structure viewed along one of six five-fold symmetry axes, exposing a central channel 2 nm in width. The overall diameter of the particle is 22 nm. <b>(B)</b> View along one of the 15 two-fold symmetry axes, exposing one of the 30 edges between two adjacent pentamers. (C) View along one of the 10 three-fold symmetry axes, exposing one of the 20 vertices at the junction between three neighboring pentamers. <b>(D)</b> Cut-open view to expose the central cavity (with the cut perpendicular to one of the five-fold axes of symmetry). <b>(E)</b> Unsharpened, unfiltered, unmasked version of the 6-Å cryo-EM structure to show the peripheral “fuzz” interpreted as glycans. <b>(F)</b> A 5.8-Å cryo-EM structure independently obtained from the same dataset. In this case, over-fitting of noise was accepted to avoid the loss of the putative carbohydrate side chains. (EM-DB ID of the 6-Å cryo-EM structure of the <i>Bg</i>AChBP dodecahedron: EMD-2055).</p