Molecular simulation reveals that pathogenic mutations in BTB/ANK domains of Arabidopsis thaliana NPR1 circumscribe the EDS1-mediated immune regulation

The NPR1 (nonexpressor of pathogenesis-related genes 1) is a key regulator of the salicylic-acid-mediated immune response caused by pathogens in Arabidopsis thaliana. Mutations C150Y and H334Y in the BTB/ANK domains of NPR1 inhibit the defense response, and transcriptional co-activity with enhanced disease susceptibility 1 (EDS1) has been revealed experimentally. This study examined the conformational changes and reduced NPR1–EDS1 interaction upon mutation using a molecular dynamics simulation. Initially, ^BTBC150Y_NPR1 and ^ANKH334Y_NPR1 were categorized as pathological mutations rather than others based on sequence conservation. A distant ortholog was used to map the common residues shared among the wild-type because the mutations were highly conserved. Overall, 179 of 373 residues were determining the secondary structures and fold versatility of conformations. In addition, the mutational hotspots Cys150, Asp152, Glu153, Cys155, His157, Cys160, His334, Arg339 and Lys370 were crucial for oligomer-to-monomer exchange. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural displacements engaging in the N-termini _α5133-178_α7 linker connecting the central ANK regions (_α13260–290_α14 and _α18320–390_α22), where prominent long helices (α5₁₆) and short helices (α3₁₀) replaced with β-turns and loops disrupting hydrogen bonds and salt bridges in both mutants implicating functional regulation and activation. Furthermore, the mutation repositions the intact stability of multiple regions (_L13C149-N356_α20^BTB/ANK-_α17W301-E357_α21^{N−ter/coiled-coil}) compromising a dynamic interaction of NPR1–EDS1. By unveiling the transitions between the distinct functions of mutational perception, this study paves the way for future investigation to orchestrate additive host-adapted transcriptional reprogramming that controls defense-related regulatory mechanisms of NPR1s in plants.