The cofactor-binding pocket.

<p>(A) A surface representation of EhMeth shows the cofactor-binding pocket in the large domain of the protein. The binding of AdoHcy in the pocket is mediated by a variety of polar and non-polar interactions shown in the close-up view. A |F_O−F_C| simulated annealing omit map contoured at 1.5 σ (blue mesh) supports the conformation of the ligand. AdoHcy is shown in a green while the interacting residues are shown in a grey ball and stick representation. (B) A LIGPLOT diagram shows the 2-dimensional projection of the protein residues interacting with the ligand. Distances between individual interacting atoms are indicated.</p

Data Collection and Refinement Statistics.

<p>Values in parentheses indicate the specific values in the highest resolution shell.</p><p>R = Σ(||F_obs|−scale |F_model||)/Σ(|F_obs|).</p><p>R_merge = Σ_hklΣ<i>_i</i>|I<i>_i</i>(hkl)−_i(hkl)>|/Σ_hklΣ<i>_i</i>_i(hkl)>, where the sum <i>i</i> is over all separate measurements of the unique reflection hkl.</p><p>R_free as R-factor, but summed over a 5.93% test set of reflections.</p

The crystal structure of EhMeth.

<p>(A) Secondary structure representation: α-helices are shown in blue, β-strands in red while loop regions are depicted in grey. Clearly the active site helix is protruding from the large domain. (B) The main-chain of EhMeth is shown in relation to its B-factor. Increased B-factors indicate a higher flexibility of the respective area of the structure, which can be observed in the active site helix and some loop areas in the small domain. (C) Surface charge representation: the proximal side of EhMeth is predominantly positively charged (blue), while the distal side of EhMeth mainly displays neutral and negative surface charges (grey, red). On the positively charged surface – the putative DNA/RNA binding area – also the cofactor-binding pocket can be seen (AdoHcy is shown in green). A dashed circle highlights a highly negatively charged pocket on the distal side of EhMeth.</p

Electrophoretic mobility shift assay with EhMeth.

<p>Different nucleic acid substrates (full length tRNA, 17 nucleotide anticodon stem-loop and DNA) were subjected to increasing concentrations of EhMeth. Arrows highlight the position of free RNA/DNA bands in comparison to the tRNA bound to EhMeth. In contrast to full length tRNA, which is clearly shifted to a distinct band, the anticodon stem loop as well as DNA are only slightly shifted and smearing upon addition of EhMeth.</p

Conservation among DNMT2 enzymes.

<p>(A) Conserved residues among DNMT2 MTases were mapped to the surface of EhMeth. Purple color displays high conservation, cyan color displays high variance. Highly conserved areas can be seen in the active site, the putative DNA/tRNA binding site as well as in a highly acidic pocket on the distal site of EhMeth. (B) A superposition of the conserved areas with the surface of M.<i>Hha</i>I (closed conformation, brown) shows that only minor divergence can be observed at the proximal site while in particular the acidic pocket on the distal side is covered – that is not existent in M.<i>Hha</i>I. The close-up illustrates that the acidic pocket is occupied by strand β10.</p

Model of the EhMeth-DNA complex.

<p>(A) Superposition of EhMeth (grey) and M.<i>Hha</i>I in closed conformation (brown) and open conformation (yellow). The superposition clearly illustrates that the active site loop of EhMeth adopts a conformation between the open and the closed conformation of M.<i>Hha</i>I. (B) Due to the high structural homology between EhMeth and M.<i>Hha</i>I a DNA-binding model could be derived from superposition with the substrate bound M.<i>Hha</i>I-structure. The M.<i>Hha</i>I-DNA neatly fits into the putative DNA-binding site of EhMeth. (C) The close-up of the active site illustrates that the flipped out cytosine is in a conformation that would allow for methyl-group transfer further supporting, that EhMeth follows the same reaction mechanism as M.<i>Hha</i>I and DNMT2.</p

L'ENMG sur INTERNET

<p>Classification of S-nitrosylated proteins in <i>Entamoeba histolytica</i> according to their biological role: Translation.</p

Analysis of <i>S</i>-nitrosylated proteins in <i>E. histolytica</i> after resin-assisted capture.

<p>A. Viability of <i>E.histolytica</i> trophozoites which were exposed to different concentrations of S-nitrosocysteine (CysNO) for 20 minutes. Data are expressed as the mean and standard deviation of three independent experiments that were repeated twice. <i>E.histolytica</i> trophozoites strain HM-1:IMSS were treated with 500 µM CysNO for 20 minutes. The protein <i>S</i>-nitrosothiols (SNO) in the cell lysates was subjected to resin-assisted capture (RAC) in the presence of 40 mM ascorbate (+ASC) or the absence of ascorbate (–ASC). B. Coomassie blue staining of <i>S</i>-nitrolysated proteins. C. Functional categories of all <i>S</i>-nitrosylated proteins. <i>S</i>-nitrosylated proteins in <i>E.histolytica</i> were classified according to their biological role. D. Confirmation of <i>S</i>-nitrosylation of three proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and the heavy subunit of Gal/GalNAc lectin after resin-assisted capture by western blotting. This figure displays a representative result from two independent experiments.</p

Proteomic Identification of <i>S</i>-Nitrosylated Proteins in the Parasite <i>Entamoeba histolytica</i> by Resin-Assisted Capture: Insights into the Regulation of the Gal/GalNAc Lectin by Nitric Oxide

<div><p><i>Entamoeba histolytica</i> is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that <i>E. histolytica</i> encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on <i>S</i>-nitrosylated proteins in <i>E.histolytica</i> is lacking. In order to fill this knowledge gap, we performed a large-scale detection of <i>S</i>-nitrosylated (SNO) proteins in <i>E.histolytica</i> trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the <i>S</i>-nitrosylated proteins that were enriched by SNO-RAC. We also found that the <i>S</i>-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of <i>E.histolytica</i> adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced <i>E.histolytica</i> adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in <i>E.histolytica</i>.</p></div

Classification of S-nitrosylated proteins in <i>Entamoeba histolytica</i> according to their biological role: Super-family of small GTPase.

<p>Classification of S-nitrosylated proteins in <i>Entamoeba histolytica</i> according to their biological role: Super-family of small GTPase.</p