Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila

Abstract

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.

The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters

“…Deletion of the second ZnF array (Kipferl Δ2nd-array ) instead had only mild impacts. The N-terminal ZAD, which is characteristic for the >90 ZAD-ZnF family members in D. melanogaster , promotes anti- parallel homodimerization but has not been directly linked to DNA binding (Jauch et al, 2003, Bonchuk et al, 2021). Yeast two hybrid experiments confirmed the dimerization capability of Kipferl’s ZAD (Fig.…”

Structures and biological functions of zinc finger proteins and their roles in hepatocellular carcinoma

“…In addition, few proteins may have several special names depending on their domain. As can be seen from the NCBI gene database, the human ZSCAN (zinc finger and scan) transcription factor family members have a uniform name ranging from ZSCAN1 to ZSCAN54 [ 33 ].…”

SafeguardingDrosophilafemale germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein

“…CG4936, which we name Identity crisis (IDC), is a 521 amino acid (aa) protein that contains an N-terminal ZAD (zinc finger-associated domain, 22-95 aa), an unstructured linker region and a C-terminal domain that includes an array of 5 C2H2 zinc fingers (386-491 aa). Studies of other ZAD-ZNF proteins suggest that the ZAD mediates protein-protein interactions and the C2H2 zinc fingers bind DNA (Bonchuk et al, 2021; Bonchuk et al, 2022; Jauch et al, 2003; Maksimenko et al, 2020; Zolotarev et al, 2016). We, therefore, hypothesize that IDC is required for phf7 repression.…”