<em>In Silico</em> characterization of OsBBX13 transcription factor and its interaction with G-Box element in rice

Arabidopsis (Arabidopsis thaliana (L.) Heynh.) BBX transcription factor AtBBX21 plays a crucial role in light, hormones, and abiotic stress signaling. It has two B-box domains (B-box I and B-box II) at the N-terminus, and the B-box II domain is responsible for DNA-binding. AtBBX21 binds to G-box (CACGTG) or G-box-like cis-regulatory elements of the promoter region, regulating its own expression as well as that of target genes. However, the functions and regulatory mechanisms of B-box proteins in rice remain largely unexplored. In addition, the three-dimensional structures of rice BBX proteins and their interactions with G-box DNA have yet to be investigated. Since orthologs tend to retain similar function during evolution, we identified AtBBX21 ortholog in rice to investigate its structure, DNA-binding ability and function through computational approaches. A MEME motif scan indicated that B-box II domain of OsBBX13 exhibits a high level of evolutionary conservation. Additionally, the OsBBX13 promoter harbors multiple light, hormone, and abiotic stress-responsive cis-regulatory elements, suggesting its role in these regulatory pathways. The N-terminal region of OsBBX13 was modeled using I-TASSER, the model with the lowest confidence-score was chosen and model quality was evaluated. OsBBX13 – G-box docking was performed using HADDOCK 2.4. The HADDOCK server was validated by redocking the homeodomain transcription factor-G-box (PBB ID: 6RYI) complex. Furthermore, a 200 ns molecular dynamics simulation using Gromacs demonstrated that OsBBX13 stably interacts with G-box DNA over time, supporting the hypothesis that it functions similarly to AtBBX21 in transcriptional regulation. Hydrogen bonding and hydrophobic interactions were found to be key stabilizing forces in the OsBBX13 – G-box complex. Our study lays the groundwork for understanding the functions and transcriptional regulation of OsBBX13, offering valuable insights for the future improvement of agronomic traits in rice. However, Experimental validation is essential to confirm the predicted gene function and regulatory mechanisms of the OsBBX13 gene.