Structural Evolution of LEAFY Reveals DNA-Mediated Cooperativity and Dimerization Shifts at the Water-to-Land Transition

Abstract The evolution of transcription factor (TF) DNA-binding specificity is a major driver of gene regulatory innovation. Unlike most TFs, which diversify through gene duplication and neofunctionalization, the plant-specific LEAFY (LFY) TF evolved novel binding specificities without extensive duplication. Here, we combine experimental structural determination and biochemical assays to reveal how LFY’s dimerization and DNA-binding preferences shifted during the water-to-land transition. We present crystal structures of the LFY DNA-binding domain (DBD) from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA, demonstrating two distinct dimerization mechanisms: one mediated by direct protein-protein interactions and another driven by DNA-mediated cooperativity. In the ancestral state, LFY likely bound DNA as a dimer through DNA-mediated cooperativity, with protein-protein dimerization emerging later, enforcing new DNA-binding preferences. Our findings support a revised evolutionary scenario for LFY, highlighting the dynamic interplay between protein-DNA and protein-protein interactions as key drivers of TF binding specificity. This work deepens our understanding of how structural adaptations in TFs underpin evolutionary transitions in gene regulation. Competing Interest Statement The authors have declared no competing interest. Data availability Structural data are deposited in the Protein Data Bank under accession codes 8S8Q, 9FWY, and 9G19. Oligonucleotide sequences are listed in Table S1. Raw data (e.g., EMSA gels) are available from the corresponding author upon request.