In recent years, several research institutes and pharmaceutical companies have achieved fundamental successes in inhibiting or reducing TLR signaling and associated effector mechanisms by using novel inhibitors. These inhibitory molecules include antibodies against TLRs, TLR-derived transmembrane (TM) peptides, bacterial-secreted proteins, and natural or synthetic small molecules, peptides, and proteins. Antagonist developers generally target the TLR ectodomain to block receptor activation. The TM and cytosolic Toll/IL-1 receptor domains also have regions that should be explored for the design of peptide-based and small molecule blocking agents. A number of preclinical and clinical breakthroughs may result in the availability of improved TLR immunomodulatory drugs to address important unmet medical needs.
Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)-a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2-leucine rich repeat (LRR) domain activates NF-κB signaling through a protein-protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD-CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD-CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2-LRR confer MDP recognition by hydrophobic and hydrogen bond (H-bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2-CARDa and the electronegative surface on zRIP2-CARD reinforced mostly by H-bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2-CARDa and Asp494 of zRIP2-CARD. Arg11 and Lys53 of zNOD2-CARDa and Ser498 and Glu508 of zRIP2-CARD are critical residues for CARD-CARD interaction and NOD2 signaling. The 2.7 Å H-bond between Lys104 of the linker and Glu508 of zRIP2-CARD suggests a possible role of the linker for shaping CARD-CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD-CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective.
Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved ‘Lysine’ at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. ‘Proline’ of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.
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