Comparison of three structures of Cbx3 chromodomain binding to methylated histone H3, H1 and G9a peptides.

<p>(A) Superposition of human Cbx3 chromodomain in complex with methylated histone H1 peptide (yellow), histone H3 peptide (orange), G9a peptide (cyan), Cbx3 chromodomains are colored as magenta, gray and green, respectively. (B) Superposition of histone H1 peptide (yellow), histone H3 peptide (orange). (C) Structure of Cbx3-H3K9me3 complex (magenta) was superposed to one protomer of the tetramer of Cbx3-G9aK185me3 complex (green) formed in one asymmetric unit. (D) The α helix (residues 70 to 79) of the chromodomain in the structure of Cbx3-G9aK185me3 complex (green) shifts 4.9 Å away from its counterpart in the structures of Cbx3-H3K9me3 (magenta).</p

Peptide binding specificity of human Cbx3 chromodomain.

*<p>N/B: No binding was detected.</p

X-ray Data collection and refinement statistics.

a<p>The values in parentheses refer to statistics in the highest shell.</p>b<p>Rmerge = |Ii−<i>|/|Ii| where Ii is the intensity of the ith measurement, and <i>is the mean intensity for that reflection.</i></i></p><i><i>c<p>Rwork = Σh|Fo(h)−Fc(h)|/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.</p>d<p>Rfree was calculated with 10% of the reflections in the test set.</p>e<p>Categories were defined by MolProbity.</p></i></i

Human Cbx3 chromodomain binds to methylated histone H1K26 and G9aK185.

<p>ITC data for Cbx3 chromodomain binding to (A) H1K26 peptides (residues 18–29) and (B) G9aK185 peptides (residues 179–190). Lower panel show fit to a one-site binding model to the binding isotherms.</p

Structure basis for Cbx3 binding to methylated histone H1K26 and G9aK185 peptide.

<p>(A and C) Electrostatic surface depiction of human Cbx3-histone H1K26me2, and Cbx3-G9aK185me3 complex. Peptide substrates are shown in a stick mode. Surfaces with positive electrostatic potential are blue, and negative potential are red. The side chain of residue H1A24 (G9aA183) inserts into the small hydrophobic pocket formed by Phe48 and Leu49 of human Cbx3. The size of the pocket is only sufficient to accommodate a methyl group but not other residue side chains. (B and D) Binding of histone H1 peptide and G9a peptide in the binding groove of Cbx3 chromodomain, respectively. Hydrogen-bonds are shown as dashed lines. Yellow: histone H1 peptide; Gray: Cbx3 chromodomain in Cbx3-histone H1K26me2 complex. Cyan: G9a peptide; Green: Cbx3 chromodomain in Cbx3-G9aK185me3 complex.</p

Surface representation of TDRD3 and SMN.

<p>(A) TDRD3 (B) SMN (PDB: 1MHN). The ligand is superimposed from the TDRD3-PG4 structure.</p

Structural comparison between human SUV39H1 chromodomain and other chromodomains, which are all colored in cyan A: human SUV39H1 chromodomain (PDB ID: 3MTS).

<p>B: human MPP8 chromodomain (PDB ID: 3R93) C: <i>Drosophila</i> HP1 chromodomain (PDB ID: 1KNE).</p

Crystal structures of TDRD3 with methyl-arginine mimics.

<p>(A) TDRD3 in complex with tetraethylene glycol (PG4). (B) TDRD3 in complex with isopropanol. The aromatic cage residues and small molecules are displayed in a stick model. (C) Superposition of the crystal structures of TDRD3 and SMN (PDB: 1MHN). The tetraethylene glycol molecule is shown in a stick model. SMN is colored in blue and TDRD3 is colored in green.</p

Fluorescence polarization binding curves of TDRD3 (aa 520–633), SMN (aa 80–170), and SPF30 (aa 65–150) to the PIWIL1-R4 peptides.

<p>The buffer used in the fluorescence polarization assay is 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT and 0.01% Triton X-100. The data are measured at 25°C and corrected for background by subtracting the free-labeled peptide background. The Kd values are the average of three independent measurements.</p

Kinetic analysis of interactions between SUV39H1 chromodomain and H3K9me3/2/1/0 or H3K27me3 peptide was performed by fluorescence polarization assay.

<p>Diagrams of different H3 peptides are indicated by different symbols. A: Kinetic analysis of interactions between truncated SUV39H1 chromodomain (aa 44–106) and H3K9me3/2/1/0 peptide. B: Kinetic analysis of interactions between complete SUV39H1 chromodomain (aa 42–100) and H3K9me3/2/1/0 or H3K27me3 peptide.</p