Computational prediction and experimental demonstration of dimerization and polymerization in the ZBTB protein family

The ZBTB family of transcription factor proteins, encoded by 49 genes in the human genome, play diverse roles in cellular processes such as chromatin structure regulation, stem cell differentiation, cellular oxidative phosphorylation, and the p53 pathway. This study focuses on unraveling the heterodimerization potential of ZBTB proteins, specifically emphasizing their N-terminal BTB domains, which facilitate inter-molecular interactions and dimerization. Despite the presence of heterodimerization within the family, homodimerization is more prevalent, and the functional significance of heterodimer formation remains unclear. To explore heterodimer formation, understand their roles, and discover novel interactions, computational predictions using AlphaFold were employed. Our studies identified ZBTB9 BTB as a noteworthy candidate with high potential for heterodimer formation. Experimental approaches testing ZBTB9 BTB domain dimerization unexpectedly identified its capacity to form biomolecular condensates. By utilizing computational structural predictions, amino acid motif discovery, and experimental mutations, we revealed that condensate formation, observed not only in the ZBTB9 BTB domain but also in ZBTB5, ZBTB6, and ZBTB8A, is linked to a conserved structural motif that mediates polymerization. We propose that 20 out of the 49 ZBTB proteins can potentially form condensates through BTB domain polymerization, involving a newly identified amino acid motif and a critical serine residue forming hydrogen bonds with alternative BTB monomers. This research contributed to the comprehension of ZBTB protein behavior, especially in the context of novel modes of interaction within the family. This study revealed unknown functions of some ZBTB protein family members.