Structure and topology of wild-type and mutant zebrafish Ig-like domains belonging to the α-DG C-terminal region.

<p>The secondary structure elements (panel A) are named according to Harpaz and Chothia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103866#pone.0103866-Harpaz1" target="_blank">[46]</a>. The β-strands are colored according to the sheet to which they belong and the N and C termini are indicated. The topology diagram of the domains is shown in panel B; β-strands are shown as circles and the small helix as a triangle.</p

Statistical support for the predicted structural models obtained from I-TASSER.

a<p>Calculated with respect to the closest structure in the PDB (1U2C).</p

Cα-RMSF values averaged per each residue over the last 30 ns of MD trajectory.

<p>Wild-type (black) and V567D (red) zebrafish simulations are shown in panel A; wild-type (green) and I591D (light blue) murine simulations are shown in panel B. Only the protein region spanning the Ig-like domain is shown.</p

Multiple alignment of dystroglycan amino acid sequences obtained using the ClustalW software

<p><b>Copyright information:</b></p><p>Taken from "Duplication of the dystroglycan gene in most branches of teleost fish"</p><p>http://www.biomedcentral.com/1471-2199/8/34</p><p>BMC Molecular Biology 2007;8():34-34.</p><p>Published online 17 May 2007</p><p>PMCID:PMC1885269.</p><p></p> The DG protein sequences from , , and are the conceptual translations of genomic available DNA sequences. Identical residues are highlighted in yellow. The cyan highlighting identifies the first intron insertion site and the red highlighting identifies the insertion site of the mini-intron. It should be noted that due to some possible sequencing mistakes, the 3' end of , and therfore the corresponding C-terminal amino acid sequence, is not fully available in the Ensembl databank. The α/β cleavage site is also highlighted (black) while the green highlighting identifies the β-DG binding epitope and the cyan one the α-DG binding epitope [39,40]. The regions chosen for designing the two primers (FISH_ext_s and FISH_ext_as) used for the DG-homologous cloning experiment in are indicated by red arrows

Amino acid sequence alignment of zebrafish and murine Ig-like domains belonging to the α-DG C-terminal region.

<p>The alignment reflects the equivalence of residues in the two structures. At the top is shown the location of the strands predicted by our molecular model of murine α-DG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103866#pone.0103866-DeRosa1" target="_blank">[17]</a>. The positions of point mutations (V567D and I591D for zebrafish and murine DG, respectively) are shown in red.</p

Backbone hydrogen bonds along the simulation trajectories for the four models.

<p>Shown is the number of backbone hydrogen bonds formed between the A′ and the G strands of zebrafish (panel A) and murine (panel B) α-DG Ig-like domains. The black and gray lines show the trajectories for wild-type and mutant systems, respectively.</p

Evaluation of I-TASSER models by using PROCHECK, ProSA-Web and VERIFY3D protein structure evaluation tools.

a<p>Ramachandran plot qualities show the percentage (%) of the residues belonging to the favoured (core), additionally allowed (allowed), generously allowed (general), disallowed region of the plot.</p>b<p>Percentage (%) of the residues with compatibility score above zero.</p>c<p>Data from De Rosa et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103866#pone.0103866-DeRosa1" target="_blank">[17]</a>.</p

Recombinant expression of α-DG(485–630)I591D.

<p>The recombinant murine mutant α-DG(485–630)I591D as well as its wild-type counterpart were purified by affinity chromatography. The fractions collected after each purification step were run on the same SDS-PAGE: lane 1: total protein extract from <i>E. coli</i> cells expressing 6xHis-Trx-α-DG(485–630)I591D; lane 2: purified 6xHis-Trx-α-DG(485–630)I591D; lane 3: 6xHis-Trx-α-DG(485–630)I591D upon thrombin cleavage; lane 4: purified α-DG(485–630)I591D (arrow); lane 5: purified wild-type α-DG(485–630); lane 6: 6xHis-Trx-α-DG(485–630) upon thrombin cleavage; lane 7: purified 6xHis-Trx-α-DG(485–630); lane 8: total protein extract from <i>E. coli</i> cells expressing wild-type 6xHis-Trx-α-DG(485–630); lane 9: protein markers.</p

Time evolution of the secondary structural elements along the MD simulation generated by DSSP.

<p>Wild-type zebrafish (panel A); V567D zebrafish (panel B); wild-type murine (panel C); I591D murine (panel D). The X-axis represents the MD trajectory time (in ns), while the residue numbers are shown on the Y-axis. Only the protein region spanning the Ig-like domain is shown.</p

Distance analysis between the A′–G and the B–G strands.

<p>Time evolution of the distances between Cα atoms of zebrafish residue pairs 481–567 (panel A), 483–567 (panel C), 489–567 (panel E) and 491–567 (panel G) and of murine residue pairs 504–591 (panel B), 506–591 (panel D), 512–591 (panel F) and 514–591 (panel H). The black and gray lines show the trajectories for wild-type and mutant systems, respectively.</p