Molecular and functional characterization of spotted snakehead NOD1 with an emphasis on structural insights into iE-DAP binding motifs employing advanced bioinformatic tools

Abstract

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are cytosolic receptors implicated in recognition of intracellular pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Depending upon their effector binding domain (EBD) at the C-terminal, the NLRs are categorized into NLRA, NLRB, NLRC, NLRP and NLRX. NOD1 is a pivotal player in immune responses against bacterial and viral invasions and interacts with pathogens via C-terminal leucine rich repeat (LRR) domain. This study aims at characterizing NOD1 in an economically important teleost of the Indian subcontinent, spotted snakehead Channa punctata. The understanding of pathogen-receptor interaction in teleosts is still obscure. In light of this, combinatorial approach involving protein modeling, docking, MD simulation and binding free energy calculation were employed to identify key motifs involved in binding iE-DAP. In silico analysis revealed that NOD1 consists of 943 amino acids comprising of one caspase recruitment domain (CARD) at N-terminal, one central NACHT domain and nine leucine rich repeat (LRR) regions at C-terminal. Structural dynamics study showed that the C-terminal β-sheet LRR4-7 region is involved in iE-DAP binding. NOD1 was ubiquitously and constitutively expressed in all tissues studied. Differential expression profile of NOD1 induced by Aeromonas hydrophila infection was also investigated. Lymphoid organs and phagocytes of infected spotted snakehead showed significant downregulation of NOD1 expression. The current study thus gives an insight into structural and functional dynamics of NOD1 which might have future prospect for structure-based drug designing in teleosts.

Communicated by Ramaswamy H. Sarma

Keywords:

• NOD1
• innate immunity
• 3D modelling
• receptor-ligand interaction
• MD simulation

Acknowledgements

Authors would like to thank Prof. Aparna Dixit (Jawaharlal Nehru University, India) for providing Aeromonas hydrophila EUS112, Prof. Shweta Saran (Jawaharlal Nehru University, India) and Strikingly Science for computational facilities, Dr. Puneet Kumar Singh for providing assistance in MD simulation and MM-PBSA energy calculations, and Dr. Ruchika Bhatt (Indian Institute of Technology, Delhi) for her guidance in molecular docking.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical statement

Institutional Animal Ethics Committee (IAEC), University of Delhi, India approved the protocol for experimental use of spotted snakehead (DU/ZOOL/IAEC-R/2018/19).

Additional information

Funding

BR & UR received financial support from Department of Biotechnology, Ministry of Science and Technology, India (Ref No. BT/PR19619/BIC/101/57/2016). Senior Research Fellowship (UGC Ref. No. 19/06/2016(i)EU-V) from University Grants Commission (UGC), India was received by BC as financial assistance. The funding agencies have no contributions in study design, data collection and analysis, preparation of the manuscript or decision to publish.