by Jitendra Maharana, Aritra Bej, Debasish Biswal, Debashis Panda, Arjun SharmaNOD1 and NOD2, founding members of the NOD-like receptor (NLR) family, play a crucial role in host defense against bacterial infections. Recognition of peptidoglycan-derived ligands triggers ATP-dependent oligomerization of the NACHT domain, exposing the CARD domains that recruit the adaptor protein RIP2 via CARD-CARD interactions to activate the NF-κB signaling cascade. Although NOD1/2-RIP2 interactions and RIP2CARD filament assembly are established, the precise interfaces that stabilize hetero-CARD filaments remain poorly defined. Here, we integrate in silico structural modeling with molecular dynamics (MD) simulations to elucidate structurally compatible arrangements of NOD1–RIP2 and NOD2–RIP2 hetero-CARD filaments. Our results reveal that NOD1CARD subunits form a structurally compatible homomeric scaffold via canonical (type-I–III) interfaces, accommodating multiple tiers of RIP2CARD rings at both filament termini. Meanwhile, the NOD2 tandem CARDs adopt multiple discrete conformations, reflecting a more intricate structural mechanism. In stable filament conformations, tandem CARDs converge at the type-II interface, with RIP2CARD rings stacking onto CARDa (top-down) and CARDb (bottom-up) interfaces, highlighting the structural role of NOD2CARDb in RIP2-mediated CARD-CARD interaction. In silico mutagenesis, involving charge-reversal and alanine scanning at key interfacial residues, disrupts NOD1–RIP2 and NOD2–RIP2 interactions at both top-down and bottom-up interfaces, leading to rapid interface destabilization within 0.1–0.4 μs of simulation. Together, these results reveal conserved and receptor-specific structural mechanisms governing NOD1/2–RIP2 CARD–CARD interactions and provide deeper structural and dynamic insights into the complex structural mechanisms for NLR-mediated inflammatory signaling.