Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita

Abstract

In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

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

“…The LRR prediction using SMART, Pfam, and InterProscan indicates five LRR motifs in zNOD2. However, multiple sequence alignment (Figure ) reveals additional four LRR motifs in zNOD2 (Maharana et al ., ). The zRIP2–CARD (471–551 aa) is present on the C‐terminal end of zRIP2, identified as described earlier.…”

“…The MDP recognition is well characterized in human (Girardin et al ., ; Tanabe et al ., ; Mo et al ., ), but in fish, it is poorly understood. Our recent in silico study provided some crucial insights into the binding modes of MDP with rNOD2–LRR (Maharana et al ., ), which allowed us to define the active site for zNOD2–LRR in the present study. The modeled zNOD2–LRR contains a concave β‐sheet rich region and a convex α‐helical region (Figure ).…”

“…Kollman charges were assigned to zNOD2-LRR, while Geister partial charges were applied to MDP using Autodock Tools 1.5. The binding site of zNOD2-LRR was defined based on the previous studies (Tanabe et al, 2004;Maharana et al, 2013). Three separate dockings were performed with grid boxes of 60 × 60 × 60 (complex I), 70 × 70 × 70 (complex II), and 80 × 80 × 80 (complex III) grid points with a grid spacing of 0.375 Å.…”

“…More recently, Grimes et al and Mo et al have described the interaction between MDP and human NOD2-LRR (hNOD2-LRR) (Grimes et al, 2010;Mo et al, 2012). Our previous computational study showed that the amino acids Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901, and Trp931 spanning β-sheet region of rohu NOD2 LRR (rNOD2-LRR) domain were essential for MDP binding (Maharana et al, 2013). The biophysical and experimental characterizations followed by site directed mutagenesis studies provided a comprehensive overview of intermolecular interaction between hNOD2-CARDab and hRIP2-CARD (Fridh and Rittinger, 2012).…”

Mutual Regulation of NOD2 and RIG-I in Zebrafish Provides Insights into the Coordination between Innate Antibacterial and Antiviral Signaling Pathways

“…Teleosts were used in several studies to investigate the structures, signaling pathways, and antipathogen functions of NOD2 and RIG-I [23,24,25,26,27,28,29,30,31,32,33,34,35]. Extensive related studies have been conducted using zebrafish, and the results indicated that Dr NOD2 and Dr RIG-I were highly conserved and similar to their mammalian counterparts [36,37,38].…”