P-264 - Cracking the Code: Structure and Dynamics of WRKY Transcription Factors Binding W-boxes in Rice

WRKY transcription factors (TFs) regulate crucial development and stress response genes in rice by binding to W-boxes (TTGACC/T) in promoters. Conventional methods for studying these interactions in planta, such as ChIP-Seq and EMSA, are often time-consuming and resource intensive. Computational modeling techniques and molecular dynamic (MD) simulation tools offer faster and more adaptable approaches to studying how various WRKY families bind to W-boxes and how mutations in W-boxes affect the interactions. We applied structure prediction tools - Alphafold3, Chai1, and Protenix servers - to predict the complexes of three different types of WRKY TFs (Group I, II, and III) interacting with a rice gene (OsOXO4) promoter that contains a W-box, and a Zn ion that is essential for complex stability. Protenix models showed higher confidence based on pLDDT ( >80), pTM ( >0.70), and ipTM ( >0.90) scores. Our structural models revealed that variations in conserved amino acids among different WRKY TF groups affected the dynamics of the binding with the W-box. Future work will include MD simulation using GROMACs to assess the stability, affinity, and interaction strength of these models based on parameters, such as root mean square deviation and fluctuations (RMSD and RMSF), hydrogen bonds, radius of gyration and solvent accessible surface area. This research provides novel structural insights into WRKY–DNA interactions and identifies key WRKY TFs involved in rice stress responses, with direct applications for rapid TF screening and breeding programs targeting enhanced stress tolerance.