Inherent backbone dynamics fine-tune the functional plasticity of Tudor domains.

Tudor domains are crucial for mediating a diversity of protein-protein or protein-DNA interactions involved in nucleic acid metabolism. Using solution NMR spectroscopy, we assess the comprehensive understanding of the dynamical properties of the respective Tudor domains from four different bacterial (Escherichia coli) proteins UvrD, Mfd, RfaH, and NusG involved in different aspects of bacterial transcription regulation and associated processes. These proteins are benchmarked to the canonical Tudor domain fold from the human SMN protein. The detailed analysis of protein backbone dynamics and subsequent analysis by the Lipari-Szabo model-free approach revealed subtle differences in motions of the amide-bond vector on both pico- to nanosecond and micro- to millisecond timescales. On these timescales, our comparative approach reveals the usefulness of discrete amplitudes of dynamics to discern the different functionalities for Tudor domains exhibiting promiscuous binding, including the metamorphic Tudor domain included in the study.

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development.

The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte's posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using a super-resolution microscopy, we show that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, we show that Aubergine forms homo-oligomers and using all purified Tudor domains, we demonstrate that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. Our data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their "interaction hubs", and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1.

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.