Structural insights into the MDP binding and CARD-CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach

Abstract

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.

NOD1CARD Might Be Using Multiple Interfaces for RIP2-Mediated CARD-CARD Interaction: Insights from Molecular Dynamics Simulation

“…NOD1 CARD showed an extensive negative surface and a small positively charged patch; however, RIP2 CARD portrayed several positive and negative patches over the surface (Fig 1B and 1C). Owing to the interest of this study, two potential type-I surface patches (both positive (α1 and α4; type-Ia) and negative (α2 and α3; type-Ib)) were identified on the basis of earlier reports on NOD1/NOD2-RIP2 and Ap1-C9 CARD-CARD interaction [15–20,23,24,33,34]. The binding patches including the critical/surface exposed residues (experimentally proved/predicted) were illustrated in Fig 1C–1E.…”

Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2

“…The putative binding residues for the Nod2:MDP interaction were described in the Nod2 crystal structure 23 and Nod2 models 33 . From these models, three residues were selected as critical binding residues of interest, R877, W931 and S933 (Figure 4a).…”

Distinct NOD2 polymorphism status is associated with differential outcome in hematopoietic stem cell transplantation: results from a meta-analysis

“…It has a C-terminal leucine-rich repeat region for sensing microbial products, a central nucleotide-binding domain (NACHT), and an N-terminal CARD (caspase activation and recruitment domain) [3] . In antigen-presenting cells, NOD2 expression leads to downstream activation of innate pathways of host defense [4] when triggered by its interaction with Muramyl Dipeptide (MDP), a component of gram-positive and -negative bacterial cell walls, subsequently leading to the activation of the pro-inflammatory NF-κB pathway. Several studies have identified three NOD2 SNPs (Arg702Trp, SNP8; Gly908Arg, SNP12; Leu1007fsinsC, SNP13) [5] as being associated with increased incidence and severity of aGvHD, increased TRM, and reduced Overall Survival (OS) [6,7] .…”