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

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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.

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Acknowledgments

This work was financially supported by the grant of National Agricultural Innovation Project NAIP, Indian Council of Agricultural Research (ICAR) (Project code C4-C30018). We thank Dr. A Bandyopadhyay, former NC and Dr. S Kochar, NC-comp 4, NAIP for their valuable suggestion and help. We gratefully acknowledge the Bioinformatics Resources and Applications Facility (BRAF) at the Centre for Development of Advanced Computing (C-DAC), Pune, India.

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  1. Fish Health Management Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, 751002, Odisha, India

    Jitendra Maharana, Banikalyan Swain, Bikash R. Sahoo, Madhubanti Basu & Mrinal Samanta

  2. Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India

    Manas R. Dikhit

  3. Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, 751002, Odisha, India

    Abhijit S. Mahapatra & Pallipuram Jayasankar

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  1. Jitendra Maharana
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  2. Banikalyan Swain
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  3. Bikash R. Sahoo
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  4. Manas R. Dikhit
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  5. Madhubanti Basu
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Correspondence to Mrinal Samanta.

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Jitendra Maharana and Banikalyan Swain are contributed equally to this work.

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Maharana, J., Swain, B., Sahoo, B.R. et al. Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita . Fish Physiol Biochem 39, 1007–1023 (2013). https://doi.org/10.1007/s10695-012-9758-2

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