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

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

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

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

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

Electron microscopy and SDS-PAGE of <i>Bg</i>AChBP.

<p><b>(A)</b> Negative staining EM of chromatographically purified rosette protein. 2% uranyl acetate was applied. Enlarged examples are also shown; note the peripheral protrusions (arrows). <b>(B)</b> Chromatographically enriched <i>Bg</i>AChBP (R, from “rosette protein”) under reducing (R) and non-reducing (nR) conditions. Note that in the second case, some material migrates as subunit dimers, whereas other material remains in the monomeric state. M, marker proteins. <b>(C)</b><i>Bg</i>AChBP in glycosylated (R) and deglycosylated (dR) form, indicating that the 31 and 60 kDa bands represent the glycosylated and the 25 and 50 kDa bands the deglycosylated form (arrows). Asterisk, N-glycosidase F as deduced from controls; M, marker proteins. <b>(D)</b><i>Bg</i>AChBP material extracted through binding to amorphous CaCO₃. R, <i>Bg</i>AChBP starting material; S, supernatant after extraction; E, fraction eluted from CaCO₃ by EDTA; W, wash buffer prior to EDTA extraction; M, marker proteins. Note that the wash buffer contains subunit dimers (arrow) and the slower migrating portion of the 31 kDa band, whereas the eluent contains its faster migrating portion. <b>(E)</b> Recombinant expression of <i>Bg</i>AChBP1. S, bacterial cell supernatant; L, bacterial cell lysate; F, flow-through of Ni column; W, wash buffer of Ni column; E, eluent of Ni column, rich in recombinant <i>Bg</i>AChBP1.</p

Resolution determination of the final 3D reconstruction of the <i>Bg</i>AChBP dodecahedron.

<p>The results for the density map in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g008" target="_blank">Fig. 8A–D</a> are shown. Compared to the 5.9 Å obtained by the FSC_0.5 criterion, the 5.6 Å determined with the FSC_1/2-bit criterion might be too optimistic. Therefore, this density map is further referred to as the “6-Å cryo-EM structure”.</p

Docking of the molecular model of BgAChBP1 into the 6-Å cryo-EM structure. (A)

<p>The molecular model of the dodecahedron depicted along one of the five-fold symmetry axes. The 6-Å cryo-EM structure is shown in opaque to demonstrate the fitting. <b>(B)</b> The same model, viewed along one of the two-fold symmetry axes. <b>(C)</b> Top view of a pentamer extracted from the 6-Å cryo-EM structure, exposing the C-face; the docked molecular model is shown in ball & stick mode. <b>(D)</b> The same structure but rotated 180° to expose the N-face (the view from inside the central cavity). <b>(E)</b> The same structure in side view. Note the large cavity (blue) representing one of the five ligand-binding pockets, and the gating C-loop (arrow). Also note that in the cryo-EM structure the C-loop is too short to fully embed the molecular model (red) which might be due to its flexibility. (PDB-ID of the BbAChBP1 pentamer: 4AOD).</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