Molecular recognition of muramyl dipeptide by Nod2 and its implications in Crohn's disease and innate immunity
Date
2017
Authors
Journal Title
Journal ISSN
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Publisher
University of Delaware
Abstract
The human body hosts a fluctuating population of trillions of microorganisms. In order to provide a delicate balance with this enormous bacterial presence, the innate immune system is charged with the recognition of immune triggering components. The innate immune system is comprised of a large number of receptors that work together to constantly survey the landscape of the cell for the presence of bacterial fragments. The synergy of this system is viewed as the first line of defense against invading pathogens. When there are disruptions to this intricate balance, disease susceptibility increases. One such disease that is proposed to arise from an atypical response to bacteria is Crohn’s disease (CD). CD is a debilitating, inflammatory bowel disorder for which there is no cure. CD patients suffer from a complex host of dysregulated interactions between their innate immune system and microbiome leading to bacterial dysbiosis and uncontrolled inflammation. ☐ The most predominant genetic link to the onset of CD is a mutation in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (Nod2). Nod2 responds to the presence of bacteria and stimulates the immune response. Mutations to Nod2 promote low diversity and dysbiosis in the microbiome, leading to impaired mucosal barrier function. Current treatments suppress the immune response rather than enhancing the function of this critical protein. New progress toward stabilizing Nod2 signaling through its interactions with small molecules holds potential in the development of novel CD therapeutics. ☐ There is limited biochemical and structural information about Nod2 due to the complexities involved in isolating the recombinant protein. Nod2 is a large, cysteine-rich protein that is membrane associated when activated. To overcome these challenges, a purification scheme utilizing a large solubility tag, glutathione-transferase, was employed. A well-folded, functional wild-type (WT) Nod2 was produced from E. coli expression. WT Nod2 demonstrated the ability to interact with Hsp70, a known binding partner. The human WT Nod2 was suggested to co-purify with a natural ligand from the peptidoglycan of its expression host. ☐ Nod2 responds to the presence of bacteria, specifically a fragment of the bacterial cell wall, muramyl dipeptide (MDP). The proposed site of this interaction is the leucine-rich repeat (LRR) domain of Nod2. Surface plasmon resonance (SPR) was used to investigate the interaction of the LRR domain with MDP. A functional and pure LRR domain was obtained from E. coli expression in high yield. The LRR domain binds to MDP with nM affinity. Fragment analysis of MDP revealed that both the peptide and carbohydrate portion contribute to the binding interaction with the LRR. Competition binding experiments confirmed that there is a specific binding interaction between the LRR and MDP. In addition, kinetic SPR experiments suggested that the fastest association to ligand requires the full MDP molecule. Slow dissociation times between Nod2 and ligand further validated Nod2 as a suitable “druggable” target. ☐ Important portions of the LRR for molecular recognition of MDP were determined by mutagenesis of putative binding residues. The proposed residues were mutated to alanine and the recombinant protein was assayed through SPR. A large decrease in affinity to MDP and its components was observed for single point mutations made to the active site. A homology model and ligand docking study was developed to propose the conformation of MDP in the active site that was consistent with the experimental data. ☐ Functional recombinant Nod2 protein maintains the ability to bind peptidoglycan. Control of the interaction between Nod2 and bacterial cell wall was demonstrated to be pH dependent. Nod2 exhibited low affinity for insoluble peptidoglycan at a pH above 7. The absence of small, soluble peptidoglycan fragments co-purifying with Nod2 was confirmed by HR-LC/MS. The ability to bind and release insoluble peptidoglycan can be used as a method of isolating functionally pure Nod2 protein. ☐ In this dissertation, the knowledge of the interactions between Nod2 and peptidoglycan has been greatly expanded. The development of an accessible source of recombinant Nod2 protein extends itself to greatly benefit the innate immunology community. The biochemical validation of the binding region of Nod2 allows for potent therapeutics to be designed mimicking those contacts. Lastly, the exploration of the Nod2 pH specific binding of peptidoglycan allows for the search for the natural ligand(s) of the protein.
Description
Keywords
Pure sciences, Crohn’s disease, Innate immunity, Molecular recognition, Muramyl dipeptide, Nod2