The Tramtrack group BTB protein domains are Arthropoda-specific multimerization modules

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.