Exploring the modulation of phosphorylation and SUMOylation-dependent NPR1 conformational switching on immune...

NPR1 (Nonexpressor pathogenesis-related genes 1) is regulated by multisite phosphorylation and SUMOylation, serving as a master switch for effector-triggered plant immunity through a transcriptional activator (TGA3) and repressor (WRKY70) are experimentally well studied. However, the conformational relationship between the various phosphorylation, un-phosphorylation states, and SUMOylation's role in the functional switch remains unclear. Using deep learning-based molecular modeling, docking, and multi-nanosecond simulations (totaling 2 μs) with end-state free energy calculations, we unveil how different phosphorylation states impact the dynamic stability of NPR1's four phospho-serine residues (Ser11, Ser15, Ser55, & Ser59) and binding of the TGA3-WRKY70 over SUMOylation. Results from our simulations show that the salicylic-acid induced ^P-Ser11/15NPR1-SUMO3 stabilizes helices and the flexible activation loop (_α22Lys423 - _α1Arg50 & _L35Asp467-Arg51α₅1, and Gly27_L3), thereby switching association with TGA3. The inter-helix salt-bridge formed (_L10Arg99-Glu323_α9 and _α14Glu280-Pro264_L6) between the phosphorylated NPR1-SUMO3-TGA3 engage in tight control of conformational regulation were disengaged in the unphosphorylated system. The ^P-Ser55/59NPR1-SUMO3-WRKY70 reorients itself and forms an electrostatic and hydrogen bond with Lys145_α7 - _L2Asp26, _L6Arg99 - Leu293_L18 and Lys262_L15 - Glu241_L15, _α13Val239 (α3₁₀), & _L17Leu267 keeps complex stable and quiescent compare to unphosphorylated NPR1-WRKY70. Subsequently, the essential dynamic and secondary structural analysis reveals that the phosphorylation inhibits the α5₁₆ (long helix) formation and reduces the communication space between the 460_{α25-βturn3-α30-L42}590 (NPR1) and 90_L9-L10107 (SUMO3), making the binding more suitable for TGA3 (260_βturn-L6270) and WRKY70 (230_L15-L16265) via activation loop. These findings, which reveal the atomic and structural details of the NPR1's post-translational modification, will illuminate future investigations into enhancing plant immunity.