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. 2010 Nov 2;5(11):e13559.
doi: 10.1371/journal.pone.0013559.

Structural studies of the tandem Tudor domains of fragile X mental retardation related proteins FXR1 and FXR2

Melanie A Adams-Cioaba  1 , Yahong Guo, ChuanBing Bian, Maria F Amaya, Robert Lam, Gregory A Wasney, Masoud Vedadi, Chao Xu, Jinrong Min
Affiliations

Structural studies of the tandem Tudor domains of fragile X mental retardation related proteins FXR1 and FXR2

Melanie A Adams-Cioaba et al. PLoS One. .

Abstract

Background: Expansion of the CGG trinucleotide repeat in the 5'-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs are locally translated in response to stimuli.

Methodology/principal findings: Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 Å, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9.

Conclusions: The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor domains reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. The crystal structures of FXR1 (A) and FXR2 (B) reveal a shared tandem Tudor domain architecture.
Tud 1 domains are colored in cyan and Tud2 domains in magenta. Coiled regions are indicated in grey. The residues forming the aromatic cage of Tud2 are shown as in stick representation and are colored yellow. (C) FXR1 (cyan) and FXR2 (purple) align well and reveal a conserved interdomain orientation. (D The previously determined structure of FMRP (PDB 2BDK) also comprises the tandem Tudor architecture. The coloring is as described for the FXR1 and FXR2 panels. (E) The sequence alignment of the FXR proteins. Residues are colored in agreement with the β-strands of panels A, B, and D. Residues in bold correspond to the ionic lock, underlined residues exhibit alterations in the HSQC spectra on peptide titration, and the asterisks denote strictly conserved residues.
Figure 2
Figure 2. Structural similarity of the Fragile X Tudor domains with other β-barrel proteins.
3D Structures of: (A) the DNA binding domain of the HIV-1 integrase (PDB 1IHV); (B) the Tudor domain of the PHD finger protein 19 (PDB 2E5Q); (C) the Tudor domain of the human SMN protein (PDB 1G5V). These three structures are shown in the same orientation based on superposition. (D) Crystal structure of FXR2 is shown for comparison. The first Tudor (tud1) is colored in cyan and the second Tudor (tud2) is colored in purple.
Figure 3
Figure 3. An interdomain ionic lock stabilizes the tandem Tudor architecture.
The FXR1 (A) and FXR2 (B) domains are stabilized by extensive interactions between the charged residues at this interface. While the residues are conserved in the FMRP protein (C), the NMR structure suggested a slightly different domain orientation that results in a loss of salt bridging. (D) The UHRF1 interface is also stabilized by the formation of a salt bridge. The ribbon traces are colored to correspond with Figure 1 and residues comprising the ionic lock are colored in yellow for all panels.
Figure 4
Figure 4. FXR1 and FXR2 preferentially recognizes trimethylated histone peptides.
The fluorescence polarization binding curves for FXR2 and H4K20 peptides are shown as a example.
Figure 5
Figure 5. Recognition of trimethylated lysine by the Tud2 domain of FXR2.
(A) Superposition of the HSQC spectra for the tandem Tudor domains of FXR2 in the presence (cyan) and absence (magenta) of the 1.5 molar excess H4K20me3 peptide. (B) HSQC spectra for FMRP reported in refenrence 22. (C) and (D) Specific chemical shifts corresponding to the predicted binding site for trimethylated lysine in FXR2-Tud2. (E) A model of trimethylated lysine recognition by FXR2-Tud2. Residues present in the crystal structure and that yield chemical shifts during titrations are indicated.
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