Structural alignments between (A) core region from all templates used in homology modeling, and (B) obtained model and recently available ligand bound DENV NS3_PRO structure (PDB id: 3U1I)

<p> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072402#B36" target="_blank">36</a>]. In (A), residues were colored based on their Qres factor, obtained after a STAMP structure alignment performed with the VMD Multiseq plugin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072402#B68" target="_blank">68</a>]. Color ranges from dark blue (highly conserved positions) to red (not conserved at all). In (B), crystallographic structure was colored with gray (NS3_PRO) and red (NS2B_CF); homology model was colored in lighter shades – white (NS3_PRO) and pink (NS2B_CF). In addition to the highly conserved active site residues position, the oxyanion hole was also preserved in this model (boxed detail).</p

Single- and two-dimensional RMSD plots from the MD simulation trajectory, and their representative conformers.

<p>In (A), RMSD values from the whole construct (NS3_PRO F, black line) were compared with RMSD values from the protein core only (NS3_PRO C, gray line). In (B), a two-dimensional RMSD plot was utilized to provide information about conformational families during trajectory. Color ranges from black (as out of the cut-out distance) to white (as in identical structures), with intermediate values depicted in the color scale. White stars mark the representative frames. (C) Snapshots of representative conformations of each family extracted from the clustered (two-dimensional) RMSD analysis (NS2B_CF and linker in red, NS3_PRO in gray, active site residues in green and NDL in orange).</p

Total pair interaction energies (potential plus kinetic).

<p>(A) Pair interactions from the ligand NDL <i>versus</i> protein (black line), and NDL <i>versus</i> water (gray line). (B) Average total energy of pair interactions from the ligand and each protein residue, allowing for contact points mapping.</p

Dengue and West Nile virus NS2B_CF-Gly-NS3_PRO multiple sequence alignment extracted from the protein databank files.

<p>This alignment allowed us to verify the completeness of each deposed structure, as well as to fill structural gaps, direct N- (blue box) and C-terminus (red box) regions and the cofactor itself. Green triangles indicate the catalytic triad (HIS₅₁, ASP₇₅ and SER₁₃₅); sequences are numbered accordingly to the full constructs and shaded based on sequence similarities (black for identical, dark gray for strongly similar and soft gray for weakly similar residues).</p

Pocket detection and evolution during MD simulation trajectory.

<p>Active site pocket (A) was identified by METAPOCKET and later monitored for changes in volume (B) by MD Pocket. In (A), DENV NS3_PRO is represented in gray; NS2B_CF and the glycine linker are in red. Active site residues are represented by green sticks, and the detected pocket is shown in a cyan surface. The pocket opposed to the active site, usually found in ligand bound structures (35) is depicted in a brown surface, merely for illustration. In (B), the red line is a smoothed curve of the black line, intended to clarify the breathing behavior of the active site.</p

Secondary structure timeline analysis as computed by the timeline plugin contained in VMD.

<p>In the graphic, the β-sheet components turn (T) and extended conformation (E) are represented in teal and yellow respectively; isolated bridges are in dark yellow; degrees of helix are in pink (α-helix), blue (3-10 helix) and red (π-helix); random coils are in white.</p

Individual residue mobility in principal component analysis.

<p>(A) The greatest eigenvector represents NS3_PRO C-terminal domain shifting, and the second eigenvector represents NS2B_CF C-terminal and glycine linker shifts. (B) Normal mode visualization from the first eigenvector, also revealing small motions that may be important to binding site plasticity.</p

Binding mode fluctuations during the MD simulation.

<p>Both ligand and binding site plasticity contributes to changes in the binding mode between the ligand and protein. The solvent accessible surface of an interacting residue or atom is represented by a blue halo around it. The diameter of the circle is proportional to the solvent accessible surface. Pink circles indicate residues involved in hydrogen-bond, polar or charged interactions; green circles indicates residues involved in van der Waals interactions; π-interactions are represented by orange lines; green arrows indicate hydrogen-bonds interactions with amino acid main chains; blue arrows indicate side chain hydrogen-bond interactions. Arrowhead directs towards the electron donor.</p

Proteolytic activity of the crude skin secretions of <i>L. labyrinthicus</i> (40 µg) on 9% SDS-PAGE incubated for 15 min with fluorogenic substrates.

<p>(Lanes 1 and 2) silver stained; (Lanes 3 and 4) Gly-Pro-AMC; (Lanes 5 and 6) l-Leu-AMC; (Lanes 7 and 8) l-Alanyl-l-Alanyl-l-Phe-Ala-AMC. Even numbers are boiled skin secretions incubated with the substrates. Mw: molecular weight markers.</p

Influence of protein concentration of the crude skin secretions of <i>L. labyrinthicus</i> on the proteolysis (mUF/min) of three fluorogenic substrates: Gly-Pro-AMC; l-Leu-AMC, and l-Alanyl-l-Alanyl-l-Phe-Ala-AMC.

<p>The assay was performed in 25± standard deviation (SD). mUF/min = milli units of fluorescence liberated per minute.</p