23 research outputs found
Interdomain interactions in hinge B area.
<p><b>Left:</b> HtrA<sup>S306A</sup> (1LCY) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref031" target="_blank">31</a>] and <b>Right:</b> HtrA<sup>V226K/S306A</sup> (5FHT; this work). Proteolytic domain is colored magenta, PDZ domain—blue. In the V226K mutant (<b>Right</b>) the distance 2,8Å indicates an H-bond/ionic pair Lys226<sub>(LC)PD</sub> -E425<sub>(α7)PDZ</sub>, absent in HtrA2 (<b>Left</b>). Otherwise, both structures overlap perfectly on each other, see text.</p
Human HtrA2 topology.
<p>(<b>A</b>) Protease domain: the N- and C-terminal helices, α1/α2 and α4, respectively, are omitted for clarity. The catalytic triad His, Asp, Ser and the loops are labeled according to the chymotrypsin nomenclature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref016" target="_blank">16</a>]. (<b>B</b>) PDZ domain. The carboxylate binding loop is indicated.</p
Modeling statistics of the starting structures.
<p>Modeling statistics of the starting structures.</p
The restraints used in restrained MD of 1LCY monomer.
<p>The values in the middle column are inferences affixed to tendencies given in Fig 1 B,C in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref038" target="_blank">38</a>].</p
HtrA trimers; PD-PDZ(1) in surface representation.
<p>L3 and the linker are shown as orange threads, L2, L1 and LD as blue threads. (<b>A)</b> wtHtrA2-peptide starting structure in orientation identical to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g003" target="_blank">Fig 3</a> bottom-right. (<b>B)</b> wtHtrA2-peptide after 50 ns MD. (<b>C</b>) active DegP PDB (entry 3CS0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref017" target="_blank">17</a>]) PDZ2 is omitted for clarity. In contrast to <b>A</b> and <b>B</b>, where L3 protrudes outside on the convex side of the trimer, in the active DegP and in other active HtrAs (not shown) L3 enters between PD and PDZ, onto the trimer’s concave, contributing (vide bottom) to the allosteric activation cascade: L3*-LD-L1-L2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.ref035" target="_blank">35</a>].</p
HtrA2 trimers after 50 ns MD, with reference to the starting structures: The pink PDZ (ribbons) make 3 lids to the PD (surface) of the trimer viewed perpendicular to C3 as in Fig 3.
<p><b>Left:</b> The resultant <b>wtHtrA2-ligand</b> complex. PDZ domains, in Units A, B, C, (cyan, canary and amber ribbons, respectively) have moved equatorially (budding-flower-like) relative to their starting (pink) positons, especially in units A & C thus opening the PD-PDZ interface. <b>Right:</b> The resultant <b>HtrA2</b><sup><b>S306A/V325D</b></sup> <b>double mutant-ligand</b> complex. Moving PDZ domains are colored as in the left panel. Only minor PDZ motions are seen; no opening of the PD-PDZ takes place.</p
Overview of simulation results.
<p>For a viewer having the N-terminus and PD C-terminal barrel in the foreground, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g005" target="_blank">Fig 5</a>, “cc” and “c” denote, respectively, counterclockwise and clockwise rotation of PDZ versus PD. The viewer sees this rotation roughly round an axis parallel to α4 and passing for “cc” between α4 and α7, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161526#pone.0161526.g005" target="_blank">Fig 5</a>, and; for “c” by the peptide-binding motif <sup>361</sup>YIGV<sup>364</sup>. “Δ”applies to “cc” only and refers to another measure of extent of the “cc” rotation, viz. to its associated arc at maximized radius. I179(β2) and L398(β15) roughly fit this radius tips, hence their C<sup>α</sup>-C<sup>α</sup> (vs. ~6Ǻ at the start) distance increases with “cc” rotation defining “Δ“. “i” indicates minute irregular motions.</p
Data collection and refinement statistics.
<p>Data collection and refinement statistics.</p
Size exclusion chromatography of HtrA3 protein variants.
<p>Calculated molecular mass is an average of at least three independent experiments. ΔN-HtrA3 to β-casein molar ratio was calculated for ΔN-HtrA3 trimers. All HtrA3 variants had the S305A substitution and were proteolytically inactive.</p><p>Size exclusion chromatography of HtrA3 protein variants.</p
Steady state kinetic parameters for HtrA3 protein variants with the peptide Ala(Mca)IRRVSYSF-ANB-NH<sub>2</sub> as the substrate.
<p>Steady state kinetic parameters for HtrA3 protein variants with the peptide Ala(Mca)IRRVSYSF-ANB-NH<sub>2</sub> as the substrate.</p