Insights into the interactions of RWP-RK and their targets: Role of serine and its conservation across species.

The RWP-RK domain is a key DNA-binding domain found in all NIN (Nodule Inception)/NLP (NIN-like proteins) and RKD (RWP-RK Domain Containing) transcription factors (TFs). The RWP-RK domain in NINs/NLPs contains a highly evolutionarily conserved sequence, RWPSRK, while in RKDs, the fourth serine (S) amino acid is substituted with either tyrosine (Y) or histidine (H). To regulate autoregulation of nodulation, the RWP-RK domain of NIN TF binds to the promoter region of CLE peptides but not RKDs. Therefore, investigating the protein-DNA interaction from a structural perspective is essential to understand the evolutionary significance of the serine (S) residue of the RWP-RK domain. Herein, we have modelled both the wild type (WT) and the variant RWP-RK domains containing substitutions like glutamic acid (E), tyrosine (Y), and histidine (H) and docked them with the modelled pCLE13 cis-element. Our docking results revealed that a helix-turn-helix (HTH) motif of the RWP-RK domain interacts with pCLE13. The WT HTH-DNA complex exhibited the most negative binding free energy, indicating a strong interaction, particularly hydrogen bonds acting between them. Simulation analysis of WT and variant models provided deeper insights into protein-DNA binding dynamics. The hydrogen bond occupancy percentage indicated that the fourth serine (S) residue is vital for maintaining a significant percentage of hydrogen bonds with DNA. The variants substituting this conserved serine (S) residue displayed energetic frustration upon binding to DNA and lost correlation among their residues. Overall, it suggested that serine (S) residue of the RWP-RK domain of all NINs/NLPs is crucial for appropriate protein-DNA interaction, which might be required for their biological relevance.