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.
Table 1.
Crystallographic and refinement statistics.
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.
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.
FXR1 and FXR2 preferentially recognizes trimethylated histone peptides.
The fluorescence polarization binding curves for FXR2 and H4K20 peptides are shown as a example.
Table 2.
Binding affinities of FXR1 or FXR2 Tudor domains to histone H3 or H4 methylated lysine peptides.
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.