Human Aminopeptidase A with bound glutamate in the crystallographic structure and the proposed binding mode of the inhibitor EC33 after molecular docking in the 3D-model of human APA.

<p>(A and C), S1 subsite visualized in the crystallographic structure of APA co-crystallized with the glutamate (green) (PDB ID 4KXD). <b>(</b>B and D), S1 subsite visualized in the 3D-model of human APA after molecular docking of EC33 (orange). Protein residues either blocking the visualization or interacting with ligands are represented with a transparent surface. Predicted hydrogen or metallic bonds are drawn as dashed lines. Interacting protein residues are labeled (C and D) accordingly to the human APA numeration. (E), Alignment of the human and mouse APA amino acid sequences with the sequences of other monozinc aminopeptidases from the M1 family. Residues in bold are the Arg-887 of human APA and the Arg-878 of mouse APA. APA, Aminopeptidase A; APN, Aminopeptidase N; APB, Aminopeptidase B; LTA4H, Leukotriene A4 hydrolase; TRHDE, Thyrotropin releasing degrading enzyme; IRAP, Insulin regulated aminopeptidase.</p

<i>Ki</i> values (nM) for several inhibitors with wild-type and mutated recombinant mAPAs targeting the S1 subsite in absence of calcium.

<p><i>Ki</i> values (nM) for several inhibitors with wild-type and mutated recombinant mAPAs targeting the S1 subsite in absence of calcium.</p

Hydrolysis of various substrates, GluNA, AspNA, AlaNA and LysNA, by recombinant mAPAs.

<p>Recombinant wild-type and mutated mAPAs hydrolysis efficiencies were evaluated using GluNA, AspNA, AlaNA and LysNA as a substrate in the absence (A) or presence (B) of 4 mM Ca²⁺. The data are the mean ± S.E of five to seven experiments performed in duplicate. *** <i>p</i>< 0,001; ** <i>p</i>< 0,01; * <i>p</i>< 0,05 and n.s (not significant) when compared with the value obtained for GluNA.</p

Effects of the divalent cation Ca²⁺ on recombinant wild-type and mutated mouse APAs enzymatic activities.

<p>Purified mAPAs (2 μg) were incubated at 37°C for 30 min with 0.5 mM GluNA with various concentrations of CaCl₂ in a final volume of 100 μl of 50 mM Tris-HCl buffer (pH7.4). (A), APA enzymatic activities expressed as a percentage of maximal hydrolysis velocity obtained with 4 mM Ca²⁺ for each purification <i>vs</i> log of Ca²⁺ concentration. (B), Effect of increasing Ca²⁺ concentration on APA enzymatic activities.</p

Kinetic parameters for wild type and mutated mAPAs in the absence or presence of Ca²⁺ using HGluβNA as a substrate.

<p>Kinetic parameters for wild type and mutated mAPAs in the absence or presence of Ca²⁺ using HGluβNA as a substrate.</p

Analysis of the molecular dynamics simulations trajectories reveals loss of inhibitor affinity for the mutated hAPAs and a larger active site pocket for the R887K mutant.

<p>(A), The pair interaction energies analysis between inhibitor and protein (blue) or inhibitor and solvent (red) indicate less spontaneous interactions between inhibitors and the mutant R887A when compared to wild-type hAPA. (B) Pair interaction energies analysis between residue 887 in the wild-type or mutated hAPAs and the inhibitors shows that the Arg-887 from the wild-type APA and the Lys-887 of the R887K mutant spontaneously interact with EC33 and GluPO₃H₂. (C), Pocket volume was extracted from the trajectories after removing the docked inhibitors, showing that the R887K mutant exhibits a larger binding pocket. Each point in the scatter plots correspond to the values calculated from a different frame in the MD trajectories, vertical bars illustrate the minimum and maximum values for each calculation, horizontal bars correspond to the means.</p

<i>Ki</i> values (nM) of several inhibitors with wild-type and mutated mAPAs targeting the S1 subsite in presence of calcium.

<p><i>Ki</i> values (nM) of several inhibitors with wild-type and mutated mAPAs targeting the S1 subsite in presence of calcium.</p

Expression and purification of wild-type and mutated mouse recombinant His-APAs.

<p><b>(</b>A), Transfected CHO cells stably expressing the wild-type or mutated Xpress-His-mAPA construct were fixed and immunolabeled with a mouse monoclonal anti-Xpress primary antibody, which was detected with an Alexa 488-conjugated anti-mouse secondary antibody. Immunofluorescence was monitored by confocal microscopy. The <i>bar</i> indicates 10 μm. (B and C), Purified recombinant wild-type and mutated Xpress-His-mAPA were analyzed by SDS-PAGE 4–12% and proteins were silver stained (B) or transferred into nitrocellulose membrane and subjected to Western blot analysis using mouse monoclonal anti-X-press antibody (C). The arrows indicate the monomeric form of APA.</p

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

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