Structural plasticity of the TDRD3 Tudor domain probed by a fragment screening hit

As a reader of di‐methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain‐containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di‐methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment‐based screening. These hits were further cross‐validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich‐like π–π interaction. The side chain of the conserved residue N596 swings away 3.1 Å to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53‐binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain‐containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure‐guided discovery of selective inhibitors targeting Tudor domains. Database Structural data are available in the PDB under the accession number 5YJ8. Fourteen inhibitors of the Tudor domain‐containing protein 3 (TDRD3) Tudor domain were identified using fragment screening. The crystal complex structure of the TDRD3 Tudor domain and hit 1 reveals a distinct binding mode from methylated arginine. Conformational plasticity of residues near the binding pocket was observed upon the hit binding. These small molecules provide a starting point for therapeutic targeting the TDRD3 Tudor domain.