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Results: 5

1.
Figure 2

Figure 2. The cofactor-binding pocket.. From: Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth).

(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 |FO−FC| 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.

Eike C. Schulz, et al. PLoS One. 2012;7(6):e38728.
2.
Figure 5

Figure 5. Electrophoretic mobility shift assay with EhMeth.. From: Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth).

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.

Eike C. Schulz, et al. PLoS One. 2012;7(6):e38728.
3.
Figure 4

Figure 4. Conservation among DNMT2 enzymes.. From: Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth).

(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.HhaI (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.HhaI. The close-up illustrates that the acidic pocket is occupied by strand β10.

Eike C. Schulz, et al. PLoS One. 2012;7(6):e38728.
4.
Figure 3

Figure 3. Model of the EhMeth-DNA complex.. From: Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth).

(A) Superposition of EhMeth (grey) and M.HhaI 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.HhaI. (B) Due to the high structural homology between EhMeth and M.HhaI a DNA-binding model could be derived from superposition with the substrate bound M.HhaI-structure. The M.HhaI-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.HhaI and DNMT2.

Eike C. Schulz, et al. PLoS One. 2012;7(6):e38728.
5.
Figure 1

Figure 1. The crystal structure of EhMeth.. From: Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth).

(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.

Eike C. Schulz, et al. PLoS One. 2012;7(6):e38728.

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