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. 2012;7(2):e30375.
doi: 10.1371/journal.pone.0030375. Epub 2012 Feb 17.

Crystal structure of TDRD3 and methyl-arginine binding characterization of TDRD3, SMN and SPF30

Ke Liu  1 , Yahong Guo, Haiping Liu, Chuanbing Bian, Robert Lam, Yongsong Liu, Farrell Mackenzie, Luis Alejandro Rojas, Danny Reinberg, Mark T Bedford, Rui-Ming Xu, Jinrong Min
Affiliations

Crystal structure of TDRD3 and methyl-arginine binding characterization of TDRD3, SMN and SPF30

Ke Liu et al. PLoS One. 2012.

Abstract

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Fluorescence polarization binding curves of TDRD3 (aa 520–633), SMN (aa 80–170), and SPF30 (aa 65–150) to the PIWIL1-R4 peptides.
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.
Figure 2
Figure 2. Preferential binding of TDRD3 to asymmetrical dimethylated H3R17 peptide over symmetrical dimethylated H3R17 peptide.
The ITC measurements were done in 20 mM Tris pH 8.0 and 200 mM NaCl using the same TDRD3 construct as used in Table 1. The measurements were taken at 25°C. Binding isotherms were plotted and analyzed using Origin Software (MicroCal Inc.). The ITC measurements were fit to a one-site binding model. (A) Histone H3R17me2a peptide (TGGKAPRme2aKQLATKA). (B) Histone H3R17me2s peptide (TGGKAPRme2sKQLATKA). (C) PIWIL1_R4me2a peptide (TGRme2aARARA).
Figure 3
Figure 3. Crystal structures of TDRD3 with methyl-arginine mimics.
(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.
Figure 4
Figure 4. Structural comparison of TDRD3 to SND1 and SPF30.
(A) Structure-based sequence alignment of the Tudor domain of TDRD3, SPF30 and SMN. The aromatic cage residues are denoted by black dots. Identical residues are colored in white on red background, and similar residues are colored in red. (B) Superposition of the crystal structures of TDRD3 and SND1 (PDB: 3OMC). SND1 is colored in cyan and its bound ligand PIWIL1_R4me2s peptide is shown in a stick model. (C) Superposition of the crystal structures of TDRD3 and SPF30 (PDB: 4A4F). The tetraethylene glycol molecule and methyl-arginine are shown in a stick model. SPF30 is colored in blue and TDRD3 is colored in green.
Figure 5
Figure 5. Surface representation of TDRD3 and SMN.
(A) TDRD3 (B) SMN (PDB: 1MHN). The ligand is superimposed from the TDRD3-PG4 structure.
See this image and copyright information in PMC

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