Unique Kinase Catalytic Mechanism of AceK with a Single Magnesium Ion

<div><p>Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg²⁺-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.</p></div

Wiberg bond index matrix^a.

a<p>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072048#pone-0072048-g005" target="_blank">Figure 5</a>. 1<b> </b>Mg²⁺.</p

Schematic diagram of 1 Mg²⁺ and 2 Mg²⁺ models.

<p>1<b> </b>Mg²⁺ model contains only one Mg²⁺ ion, here it specially refers to dissociative I and associative I models. The 2<b> </b>Mg²⁺ models contain two Mg²⁺ ions, which include the associative II, associative III and dissociative III models.</p

Generation of double knockout of ApoE and smooth muscle specific p53 mice on a C57BL6 background

<p>(A) Schematic of the breeding protocol to generate the desired genotypes used in this study. White boxes indicate intermediate genotypes, green boxes indicate control genotypes and pink box indicates double knockout mice. (B) PCR genotyping for three alleles. (C) Whole aorta, liver, heart and cultured smooth muscle cells from ApoE^-/- /p53^-/- (tamoxifen-treated) and ApoE^-/- /p53^flox/flox control mice (without tamoxifen) were lysed and analyzed by Western blot to confirm targeted p53 knockout in smooth muscles cells.</p

The role of p53 in PDGF-induced circular dorsal ruffle (CDR) formation and cell migration

<p>(A) Western blot of passage 2 primary VSMCs extracted from mouse aortas and Mouse Embryonic Fibroblasts (MEF), as a negative control for SM α-actin. GAPDH was used as a loading control. (B) Immune-stain of VSMCs with Cy3-SM α-actin (red) and DAPI (blue). Scale bars represent 20 μm. (C) 20,000 VSMCs cultured in a 6 well dish were counted after 2 days. Error bars represent standard deviation of 3 separate trials and * indicates p value <0.05. (D)Serum-starved VSMCs stimulated with 20 ng/ml PDGF for 10 min and stained for cortactin (green) and F-actin with TRITC-phalloidin (red) shows representative CDR formation. Scale bar represents 20 μm. (E) Serum-starved VSMCs stimulated with 20 ng/ml PDGF were time-lapse imaged for 60 min to count the percentage of cells producing CDRs. Error bars represent standard deviation from 3 separate trials. (F) Western blot of serum-starved VSMC lysates taken at various time intervals after PDGF stimulation. β-actin was used as a loading control. (G-H) VSMCs grown to 100% confluency were scratched to create a wound in the cell monolayer. 6 hours post-wound, cells were stained for F-actin with TRITC-phalloidin (red) and DAPI (blue). Initial wound width measurements were taken and compared to wound width after 6 hours and plotted as relative values. Scale bars represent 100 μm. Error bars represent standard deviation of 3 separate trials and * indicates p value <0.05.</p

Mapping of the amide proton protection factor at 17°C onto the surface of CfAFP-501.

<p>Residues with low protection factor are colored red, and residues with high protection factor in white. The left view has the orientation showing the ice-binding surface.</p

The amide proton Δδ/ΔT-CSD correlation plot of CfAFP-501.

<p>The statistical cutoff line, Δδ/ΔT  = −2.41× CSD −2.11, is drawn. The inset shows the amide proton Δδ/ΔT-CSD correlation plot for the residues of the first Thr row.</p