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) KDs 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).
