Sluchanko, N. | Search bioRxiv

bioRxiv2025

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 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 conservedCGEx motif. Yeast two-hybrid assays, SEC-MALS, 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 IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence towards the dimerization of C2H2 domains. The structure revealed that dimerization is mediated by the interface between the core C2H2 fold and the adjacent {beta}-strand containingCGEx motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed thatCGEx-type C2H2 domains are specific to Diptera. Finally, we demonstrate that putatively dimerizing C2H2 domains containing additional structural elements beyond the canonical fold are widespread among diverse eukaryotic taxa, with the highest prevalence observed in insects. These findings establish that single C2H2 domains can mediate self-association and identify theCGEx-type C2H2 domain as a distinct structural subclass specific to Dipteran insects.

molecular biology↗

bioRxiv2017

Solution Structure Of Human STARD1 Protein And Its Interaction With Fluorescently-Labeled Cholesterol Analogues With Different Position Of The NBD-Group

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 remains debatable, and the solution conformation of STARD1 is insufficiently characterized, partially due to its poor solubility. Although cholesterol binding to STARD1 was widely studied by commercially available fluorescent NBD-analogues, the effect of the NBD group position on binding remained unexplored. Here, we analyzed in detail the hydrodynamic properties and solution conformation of STARD1 and its interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different position, namely 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). The small-angle X-ray scattering (SAXS)-based modeling and docking simulations show that, apart from movements of the flexible {Omega}1-loop, STARD1 unlikely undergoes significant structural rearrangements proposed earlier as a gating mechanism for cholesterol binding. While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence yield. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP outperforms commonly used 22NC and is recommended for future studies. Our study benefits from state-of-the-art techniques and revisits the results of the STARD1 research over the last 20 years, revealing important novel information.

biochemistry↗