The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors

Abstract

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.

FIGURE 1.

Binding affinity of sDMA containing peptides of MILI to individual TDRD1 eTud domains. (A) Schematic representation of mouse TDRD1 and the N terminus of MILI. TDRD1 has an N-terminal MYND domain and four tandem extended Tudor domains. The N terminus of MILI contains multiple arginine residues being mono- and symmetrically dimethylated (R highlighted in red). The three methylated peptides used in this study are indicated. (B) K_Ds derived from ITC measurements for three sDMA-containing peptides of MILI binding to the four individual eTud domains of mouse TDRD1. Error bars indicate the standard deviation of two independent measurements. (C) Representative ITC experiments and fits to the data for the four single eTud domains (TD1, TD2, TD3, and TD4) of TDRD1 with the R74me2 peptide of MILI. The data were fitted to a single-site binding model. (D) ITC data for the double eTud domains TD2–3 and TD3–4 with the R74me2 peptide and with the R4574me2s doubly methylated peptide of MILI. The sequence of R4574me2s is indicated below the corresponding curves. The model for fitting the tandem TD2–3 and TD3–4 domains with the R74me2 ligand assumes two different binding sites as the peptide binds differently to each individual Tudor domain. For the R4574me2s peptide binding, a single-site binding model was used (one double methylated peptide to one tandem domain).

FIGURE 2.

TDRD1 TD3 structure in complex with R45me2 peptide. (A) Cartoon representation of TD3 (blue) in complex with the MILI R45me2 peptide (yellow). Secondary structure elements are labeled in white. The aromatic cage residues (orange) and the sDMA (yellow) are represented as sticks. (B) Superposition of the structures of TD3 (blue), SND1 (magenta), and eTud11 (cyan). The C-terminal helix of SND1 is absent from the other two structures. The different orientations of the distal end of the helix α2 could play a determining role in defining the orientation of the peptide. (C) The N-terminal extension (chocolate color) of TD3 makes a third connection between the Tudor core (left) and SN-like (right) subdomains against which it packs via large hydrophobic residues such as W699, W701, and F704 as shown. (D) Structure-based sequence alignment between TD3 (45r4), eTud11 (PDB code 3NTI), and SND1 (PDB code 3OMC). Residues with similarity above 70% are displayed in red.

FIGURE 3.

TD3 binds the R45me2 peptide in a different orientation from that previously observed in eTud domains–peptide complexes. (A) The sDMA in the aromatic cage of TD3 colored as in Figure 2A. The dipeptide GD stabilizing the cage and E798 interacting with N796 are also shown. (B) Details of the interactions between the R45me2 peptide and TD3. Colors are as in A with additionally residues of the SN-like domain interacting with the peptide being in magenta. Blue dashed lines indicate putative hydrogen bonds. (C) Structural comparison of the aromatic cages of TD3 (blue) and eTud11 (green) in complex with their corresponding peptides highlighting the different direction of entry of the two methylarginine groups. All other such structures exhibit the same conformation as eTud11/R13me2. (D) Diagram showing the orientation of various sDMA-containing peptides with reference to the TDRD1 TD3 structure (blue) obtained by superposition of eTud–peptide complexes of known structure. R14me2 (PDB 3OMG, green) and R4me2 (PDB 3OMC, cyan) were co-crystallized with the SND1 protein, while R15me2 (PDB 3NTI, magenta) and R13me2 (PDB 3NTH, wheat) were crystallized with eTud11 from Drosophila TUDOR. All of the above-mentioned peptides enter the aromatic pocket from the “bottom,” while the R45me2 peptide (yellow) enters from the “top” of the structure. The observed peptide residues in each structure are shown at the left with the sDMA highlighted in red. N- and C-termini of the peptides are also indicated. (E) Conserved residues in murine TDRD eTud domains. In a stick-model representation, the residues conserved in most of the eTud domains of TDRD proteins are shown (green). The absolutely conserved R775 is involved in extensive interactions with residues of the Tudor domain, thus stabilizing the aromatic cage. Blue dashed lines indicate hydrogen bonds.

FIGURE 4.

“Active” and “inactive” extended Tudor domains of TDRD1. (A) K_Ds derived from ITC measurements for the binding of isolated symmetrically dimethylated arginine (Rme2) and R45me2 peptide to individual wild-type (wt) TDRD1 TDs and N325Y, Y774N, and N796A mutants. Error bars represent SD values from two experiments. (B) Representative ITC experiments and fits to the data. The four single eTud domains (TD1, TD2, TD3, and TD4) of TDRD1 with the isolated symmetrically dimethylated arginine (Rme2). (C) Multiple sequence alignment of the four individual Tudor domains of TDRD1. The first red arrow highlights the position of N325 of TD1, and the second arrow the Y774 of the aromatic cage of TD3. Mutations on both residues are critical for binding sDMAs (see A). (D) Binding of TD2 and TD3 domains of TDRD1 to sDMA-containing peptides monitored by NMR. Each panel shows an overlay of the ¹H,¹⁵N HSQC spectra of the respective domain when free (black), when saturated with an excess of naked sDMA (red), and when saturated with MILI derived sDMA-containing peptides (green or cyan). For well-resolved peaks, chemical-shift perturbations arising from sDMA contacts are annotated with red arrows and those induced by interactions with flanking residues with black arrows in orange background. Notice that other peaks are affected by both sDMA and flanking residues but are not labeled. (E) Pull-down assays of endogenous murine proteins MILI, MIWI, and mouse Vasa homolog (MVH) by individual and multiple TDs of TDRD1. His-tagged constructs with four Tudor domains (TD1–4) and single Tudor domains (TD1, TD1 N325Y mutant TD2, TD3, TD4) were used. Size markers in kiladaltons are indicated.

FIGURE 5.

Overall architecture of the TD1–4 domains of TDRD1 as determined from small-angle scattering data. (A) TD1–4 ab initio models showing “most representative (filtered)” (gray) and “average” envelope (light blue) as given by DAMAVER. The rigid body model was produced using CORAL (Petoukhov and Svergun 2005). TD1 (salmon), TD2 (light red), and TD4 (deep blue) models were based on Tudor-SN (pdb code 2WAC). TD3 (light blue) was fitted using the crystal structure determined here. Flexible linkers connecting the eTud domains, positioned by CORAL, are depicted as spheres (olive green). (B) Ab initio model (orange) derived from the TD1-2 SAXS data. This is consistent with either half of the TD1–4 model. (C) SAXS data (black dots with error bars) and fits for TD1–4 and TD1–2 (solid lines). (Top) TD1–4 data. Fit of the ab initio model shown in A (gray) and the multidomain model created by CORAL (purple). (Bottom) TD1–2 data. Fit of the ab initio model shown in B (orange). Fits of the tandem TD1–2 and TD3–4 parts of the CORAL model are, respectively, shown in red and blue.