Simulations of ApAAP in open conformation.

<p>A) The cross-correlated motions at the interdomain interface (correlation threshold of 0.4) in simulations of open ApAAP are shown as green lines (positive correlations) and blue lines (negative correlations). The β-propeller and the catalytic domains are shown in pale-green and white, respectively, whereas the α1-helix is highlighted in pale-cyan. D376, which is the hinge residue proposed for the opening of the catalytic cleft <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035686#pone.0035686-Harmat1" target="_blank">[33]</a> is shown. B) The salt bridge networks at the interface between the β-propeller and the catalytic domains in ApAAP open conformations are shown as spheres connected by yellow/green lines according to their persistence. The β-propeller domain and the catalytic domain are highlighted in pale-green and white, respectively, whereas the α1-helix in pale-cyan. Catalytic residues are shown as sticks.</p

Salt bridge interactions at the interface between the two protein domains in ApAAP-Δ21.

<p>The salt bridge pairs are indicated by lines and the residues involved in the salt bridges and their networks as spheres. The catalytic residues are shown as sticks and the β-propeller and catalytic domain colored in marine and magenta, respectively. The salt bridges are connected by lines of different shade of colors according to their persistence in the MD ensemble (from green to blue for increasing persistence values).</p

Protein dynamics fingerprint for wt, Δ21, and mutants ApAAP variants.

<p>The projections of the displacement described by the first principal component on the 3D structure are shown for wt (A), Δ21 (B), I12A (C), V13A (D), V16A (E), L19A (F), and I20A (G) ApAAP variants with the different simulation frames colored with different shade of colors from light cyan to purple. The catalytic triad and the α1-helix are shown as spheres and cartoon, respectively. The analyses were also carried out for the second and third components, which provide the same general view and are therefore not presented here.</p

Salt bridge clusters in wild type ApAAP.

<p>Salt bridges belonging to cluster 1 (A, blue), cluster 2 (B, E, cyan) and clusters 3 (B, yellow) and 4 (B, green) are shown as spheres and connected by sticks. C–D) Details on salt bridges belonging to cluster 1 and located in proximity of the catalytic site. E) Details of some salt bridge networks located in cluster 2. The α1-helix is highlighted as cyan cartoon. The sticks connecting the salt bridges are colored according to the persistence of the interactions in the simulations (from light to dark magenta for increasing persistence values).</p

Summary of the multi-replica all-atom MD simulations.

<p>Summary of the multi-replica all-atom MD simulations.</p

Correlated motions at the interdomain interface.

<p>A) The open structure of ApAAP identified by X-ray crystallography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035686#pone.0035686-Harmat1" target="_blank">[33]</a> is shown as a reference. The observed crucial residues for mediating cross-correlated motions in the simulations of the ApAAP closed form (panel B) are shown as spheres. B) The dynamical cross-correlations at the interdomain interface (correlation threshold of 0.4) in wild type ApAAP are shown as red lines. The β-propeller and the catalytic domains are shown in pale-cyan (A)/blue (B) and pale-green (A)/white (B), respectively, whereas the α1-helix is highlighted in cyan. The hinge residue proposed for the opening of the catalytic cleft, D376 is shown in dark green (A) and black (B), respectively.</p

The shortest communication paths from the hydrophobic residues of the α1-helix to the catalytic site.

<p>The shortest and highest frequency pathways, as detected by PSN-DCCM analysis, between V16 (A), L19 (A), I20 (B) and the catalytic H556 and D524 are shown as sticks proportional to the intensity of the correlation.</p

α1 deletion perturbs the architecture of ApAAP active site.

<p>A–F) Local network of salt bridge interactions mediated by R526 in the wild type ApAAP (A), ApAAP-Δ21 (B), ApAAP-I12A (C), ApAAP-V13A (D), ApAAP-V16A or ApAAP-I19 (E), ApAAP-L20A (F) are shown with different shade of color which are proportional to the persistence of the interaction during dynamics (with the darker colors indicating an higher persistence). G) wild type ApAAP and ApAAP-Δ21 average structures from the simulations are shown in white and blue, respectively. The catalytic residues are indicated by sticks. H–I) Coupled motions of the catalytic triad. The coupled motions which involve the catalytic triad are shown for wild type ApAAP (red sticks, H) and ApAAP-Δ21 (green sticks, I). Catalytic residues are shown as sticks and the α1-helix highlighted in cyan.</p

Salt bridges at the interdomain interface in wild type ApAAP.

<p>A general view (A) and zoom on the upper and lower regions (B, C) are shown. Residues involved in salt bridges and their networks are indicated as spheres connected by lines of different shade of magenta according to their persistence in the MD ensemble (from light to dark magenta for increasing persistence values). The β-propeller domain and the catalytic domain are highlighted in marine and white, respectively, whereas the α1-helix in cyan. Catalytic residues are shown as sticks.</p

ApAAP 3D structure, <i>in silico</i> alanine scanning and hydrophobic interaction networks.

<p>A) The secondary structure elements of ApAAP and its 3D architecture are shown, with α-helices colored in different shade of colors from the N- (blue) to the C-terminal (red) extremity. The catalytic triad (S445, D524, H556) is shown as sticks and spheres. B) The residues which have been predicted to destabilize (blue; I12, V13, V16, L19, I20, V22 and K24), partially destabilize (cyan; F9, R18, D15, E23) or not influencing (pale cyan; E8, S10, R11, R14, and E17) the ApAAP 3D structure, upon <i>in silico</i> alanine mutations, by a consensus of three different programs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035686#s4" target="_blank">Materials and Methods</a>) are shown as spheres. Side chains of residues which, upon <i>in silico</i> alanine mutations, are predicted to have the most detrimental effects on protein stability (blue) are shown as sticks. Individual <b>ΔΔ</b>G values calculated by FoldX, I-Mutant and PoPMusic are reported I <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035686#pone.0035686.s005" target="_blank">Table S1</a>. C–D) The network of intramolecular hydrophobic interactions involving α1-helix residues in the X-ray structure (C) and as derived by the molecular dynamics (D) are shown as spheres connected by sticks.</p