Human FMRP contains an integral tandem Agenet (Tudor) and KH motif in the amino terminal domain

Abstract

Fragile X syndrome, a common cause of intellectual disability and autism, is due to mutational silencing of the FMR1 gene leading to the absence of its gene product, fragile X mental retardation protein (FMRP). FMRP is a selective RNA binding protein owing to two central K-homology domains and a C-terminal arginine-glycine-glycine (RGG) box. However, several properties of the FMRP amino terminus are unresolved. It has been documented for over a decade that the amino terminus has the ability to bind RNA despite having no recognizable functional motifs. Moreover, the amino terminus has recently been shown to bind chromatin and influence the DNA damage response as well as function in the presynaptic space, modulating action potential duration. We report here the amino terminal crystal structures of wild-type FMRP, and a mutant (R138Q) that disrupts the amino terminus function, containing an integral tandem Agenet and discover a novel KH motif.

Figure 1.

Structure of FMRP amino terminal domain. (A) Schematic representation of human FMRP structure. Tandem Agenet and KH0 domain, KH1 and KH2 domains and RGG box are shown. (B and C) Two views of FMRP amino terminal structure, with Agenet1 in blue, Agenet2 in green and KH0 in brown. (D) Intra-molecular polar interactions on the interface of Agenet1 (blue) and 2 (green). (E) Intra-molecular polar interactions between strand β5 of Agenet1 (blue) and Loop-2 (green). (F) Intra-molecular hydrophobic interactions on the interface of Agenet1 (blue) and KH0 (brown).

Figure 2.

Structural similarity of FMRP tandem Agenet with other tandem Tudor domains. (A) FMRP amino terminal structure with aromatic cage residues in Agenet1 (blue) and Agenet2 (green) indicated. (B–D) Tandem Tudor domains of UHRF1, SHH1 and 53BP1 bind methylated lysine in the Tudor1 aromatic cage (corresponds to Agenet1 of FMRP). (E) Tandem Tudor domain of JMJD2A binds methylated lysine in the Tudor2 aromatic cage (corresponds to Agenet2 of FMRP).

Figure 3.

FMRP KH0 motif is an integral part of the amino terminal structure. (A) Sequence and structural alignments of FMRP KH0, KH1 and KH2. White letters on black are identical or conserved residues among all three KH modules, and white on gray are identical or conserved in at least two. The G-X-X-G motifs of KH1 and KH2 and A-X-X-A motif of KH0 are depicted in white on red background. The position of the patient mutation (R138Q) and hydrophobic residues involved in the hydrophobic core (Fig. 1F) unique to KH0 are shown in red. Conserved residues are as defined by the following groupings: V, L, I and M; F, Y and W; K and R, E and D; Q and N; E and Q; D and N; S and T, and A, G and P. (B) Cartoon structure of Agenet1-Agenet2-KH0 (PDB: 4QW2) connected to KH1-KH2 (PDB: 2QND) by a flexible linker. (C and D) The surface charge distribution of amino terminal Agenet1-Agenet2-KH0 (C) and KH1-KH2 (D) at neutral pH, displayed as blue for positive, red for negative and white for neutral, in a similar orientation as shown in (B). The orange circles indicate the position of A-X-X-A (KH0) or G-X-X-G (KH1 and KH2).

Figure 4.

Comparison of WT and R138Q structures. (A) WT (gray, space group P4₃2₁2) and R138Q (brown, space group P4₁2₁2) crystal structures are highly similar with an r.m.s.d. of 0.8 Å. (B) Superposition of WT (gray) and R138Q (brown). Residues R138 and Q138 of helix αA are indicated.