Structural and dynamic basis of NOD2 tandem CARD association and NOD1/2–RIP2 signaling complexes

NOD1 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.Author summary: NOD1 and NOD2 are cytosolic immune receptors that sense bacterial peptidoglycans and start inflammation by recruiting the adaptor protein RIP2. This recruitment relies on small signaling domains called CARDs, which nucleate and assemble into filament-like polymers that activate the NF-κB pathway. Although NOD1/2 and RIP2 hetero-CARD filaments are thought to be critical for immune signaling, their structures have been challenging to resolve experimentally. As most CARD domains share a very similar structural fold, and the hetero-filaments look much like homomeric ones, cryo-EM averaging can mask subtle differences between subunits. To address this issue, we used structural modeling and molecular dynamics simulations to map the likely architectures of NOD1-RIP2 and NOD2-RIP2 hetero-CARD filaments. Our results show that NOD1 CARDs can form a stable homomeric scaffold that can accommodate RIP2CARD rings at both ends of the filament. In contrast, NOD2 contains two CARDs that adopt multiple conformations and form a structurally compatible arrangement involving a type-II interface, with the second CARD (CARDb) providing the additional binding surface for RIP2CARD. Together, these computational results reveal both shared and receptor-specific mechanisms by which NOD1 and NOD2 drive RIP2 filament formation during immune signaling.