Beyond DNA binding: single C2H2 zinc fingers with adjacent β-strands mediate dimerization in Drosophila transcription factors

C2H2 proteins, characterized by DNA-binding C2H2-type zinc finger domains, constitute the largest group of transcription factors. In addition to binding DNA, C2H2 domains can mediate protein–protein interactions, facilitating the oligomerization of C2H2 proteins. In this study, we identified eight C2H2 proteins in the Drosophila genome that feature a unique single C2H2 domain containing a conserved “CGxΦ” motif. Yeast two-hybrid assays, size-exclusion chromatography coupled with multi-angle light scattering, and chemical cross-linking experiments revealed a strong propensity of these domains to form dimers. Using NMR spectroscopy, we determined the solution structure of the dimeric C2H2 domain from the IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence for the dimerization of C2H2 domains. Dimerization is mediated by the interface between the core C2H2 fold and the adjacent β-strand containing the CGxΦ motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed that “CGxΦ”-type C2H2 domains are specific to Diptera. Finally, our predictions demonstrate that dimerizing C2H2 domains containing additional structural elements could be widespread among eukaryotic taxa, with the highest prevalence in insects. These findings establish that single C2H2 domains can mediate self-association and identify the CGxΦ-type C2H2 domains as a distinct structural subclass specific to dipteran insects. © The Author(s) 2026. Published by Oxford University Press.