Structural Basis For Recognition Of Methylarginined Proteins By Tudor Domains

Post-translational modifications (PTMs) of histones are important epigenetic modifications, which, together with DNA modifications and non-coding RNAs, form the cornerstones of chromatin biology and epigenetics. The enzymes responsible for bringing about steady-state balance of PTMs and the PTM recognition proteins, which are referred to WRITERS, ERASERS and READERS, have attracted lots of attention. By means of X-ray crystallography and NMR techniques, the molecular mechanims of histone and protein modifications (acetylation, phosphorylation and lysine methyllation) and their recogniztion have been reported. Although the enzymes responsible for arginine methylation have been known for over10years, but the binding mechanism of the methylarginine recognition is still elusive.Recently, it was reported that the Tudor domain containing proteins (TDRDs), including TDRD1to TDRD12, comprise a family of proteins which can read the methylarginine marks. Hence, here we systematically analyzed the binding specificity of the Tudor domains of the TDRD proteins against a family of methylarginine peptides, and elaborated the binding mechanism of the Tudor domain proteins by structural studies coupled with binding assays, sequence alignment, and mutagenesis.We established that, like other germ-line Tudor domain proteins, the ancestral staphylococcal nuclease domain-containing1(SND1) polypeptide is expressed and associates with PIWIL1/Miwi in germ cells in mouse. We also found that human SND1protein binds PIWIL1in an arginine methylation-dependent manner with a preference for symmetrically dimethylated arginine. In order to further study how SND1recognize methylarginine marks, we investigated the crystal structures of the human SND1extended Tudor domain in complex with symmetrically dimethylated arginine peptides PIWIL1_R4me2s and PIWIL1_R14me2s with the resolution of1.77A and1.85A, respectively. Both complex structures show that the extended Tudor domain of SND1protein binds the peptides through cation-π, hydrophobic, van der Waals, and hydrogen bond interactions. Moreover, we found that both the entire Tudor domain and a bifurcated SN domain are required for the binding, and the canonical Tudor domain alone is insufficient for methylarginine ligand binding. Our crystal structures also show that the intact SND1extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1in different orientations. This analysis explains how SND1preferentially recognizes symmetrical dimethylarginine via an aromatic cage and conserved hydrogen bonds, and provides a general paradigm for the binding mechanisms of methylarginine containing peptides by extended Tudor domains, and also provides important information for the further study of the functions of SND1and methylarginine containing proteins.TDRD3(Tudor domain containing protein3) is the only identified protein, which could bind methylarginine marks on histones. In addition, it is also found that the Tudor domain of TDRD3is highly homologous to those of the SMN and SPF30proteins. Through a series of FP and ITC experiments, we found that they preferentially bind different methylarginine marks. For example, TDRD3preferentially recognizes asymmetrical dimethylated arginine mark, while SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine, and SPF30is the weakest methylarginine binder, which only binds the GAR motif sequences. Interestingly, our data also showed that the canonical Tudor domain alone is sufficient for methylarginine ligand binding for the TDRD3and SMN protein, which is very different from the Tudor domain of SND1. Furthermore, we also reported the high-resolution crystal structures of the Tudor domain of TDRD3in complex with one small molecule PG4at1.9A, in which the small molecule PG4occupies the aromatic cage of TDRD3, mimicking the methylarginine residue.Guided by our understandings gained from the SND1and TDRD3studies, we systematically studied the binding affinities of the Tudor domains from other TDRD family members with methylarginine peptides. Our results showed that these Tudor domains exhibit distinct binding specificities. For example, TDRD1, TDRD7, TDRD9and SND1proteins preferentially bind to symmetrically dimethylated arginine PIWIL1peptides, TDRD3and TDRD4preferentially bind to asymmetrically dimethylated arginine PIWIL1peptides, and TDRKH (TDRD2) preferentially binds to unmodified PIWIL1peptides. Furthermore, we also found that only a subset of Tudor domains can recognize methylarginined peptides in the multiple Tudor domain proteins. These discoveries lay an important foundation for the futuer function study of the methylarginined marks and TDRDs.