The toll-like receptor 5 (TLR5) is the most conserved important pattern recognition receptors (PRRs) often stimulated by bacterial flagellins and plays a major role in the first-line defense against invading pathogenic bacteria and in immune homeostasis. Experimental crystallographic studies have shown that the extracellular domain (ECD) of TLR5 recognizes flagellin of bacteria and functions as a homodimer in model organism zebrafish. However, no structural information is available on TLR5 functionality in the major carp Cirrhinus mrigala (mrigala) and its interaction with bacterial flagellins. Therefore, the present study was undertaken to unravel the structural basis of TLR5-flagellin recognition in mrigala using structural homodimeric TLR5-flagellin complex of zebrafish as reference. Integrative structural modeling and molecular dynamics simulations were employed to explore the structural and mechanistic details of TLR5 recognition. Results from structural snapshots of MD simulation revealed that TLR5 consistently formed close interactions with the three helices of the D1 domain in flagellin on its lateral side mediated by several conserved amino acids. Results from the intermolecular contact analysis perfectly substantiate with the findings of per residue-free energy decomposition analysis. The differential recognition mediated by flagellin to TLR5 in mrigala involves charged residues at the interface of binding as compared to the zebrafish complex. Overall our results shows TLR5 of mrigala involved in innate immunity specifically recognized a conserved site on flagellin which advocates the scientific community to explore host-specific differences in receptor activation.

Nucleotide-binding and oligomerization domaincontaining protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2−RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa−CARDb) and their interaction with RIP2 CARD . Our results suggest that a negatively charged interface of NOD2 CARDa and positively charged type-Ia interface of NOD2 CARDb are crucial for CARDa−CARDb association and the type-Ia interface of NOD2 CARDa and type-Ib interface of RIP2 CARD predicted to be involved in 1:1 CARD−CARD interaction. Moreover, the direct interaction of NOD2 CARDb with RIP2 CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD−CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2−RIP2 association in higher and lower eukaryotes.