In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.
BTB (Bric-a-brack, Tramtrack and Broad Complex) is a diverse group of protein-protein interaction domains found within various eukaryotic proteins. In metazoans, many DNA-binding transcription factors contain a dimerizing BTB domain with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize by an unknown mechanism. Here, we demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non TTK type BTB domains. A combination of multi-angle laser light scattering (MALS), molecular modeling, small-angle X-ray scattering (SAXS), and microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers. The dimers are connected through a previously uncharacterized interface that involves the formation of a β-sheet by β-strands of neighboring dimers. Such architecture was further supported by site-directed mutagenesis. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations. According to the mutagenesis data, such associations occur mostly between different homodimers through the dimer-dimer interaction interface, which is highly similar among members of the TTK-type BTBs. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.
Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3’UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3′UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3′UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.
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