Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine

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The Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2 Mackenzie L. Lauro, Elizabeth A. D'Ambrosio, Brian J Bahnson, and Catherine Leimkuhler Grimes ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00154 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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ACS Infectious Diseases

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

Mackenzie L. Lauro, Elizabeth A. D’Ambrosio, Brian J. Bahnson, and Catherine L. Grimes*

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716

* Corresponding author email: [email protected]

Keywords: Nucleotide-binding oligomerization domain-containing 2, muramyl dipeptide, peptidoglycan, innate immunity, leucine-rich repeat domain, surface plasmon resonance

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Abstract Genetic mutations in the innate immune receptor nucleotide-binding oligomerization domaincontaining 2 (Nod2) have demonstrated increased susceptibility to Crohn’s disease, an inflammatory bowel disease which is hypothesized to be accompanied by changes in the gut microbiota. 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. Surface plasmon resonance and molecular modeling were 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 high affinity, with a KD of 212 ± 24 nM. Critical portions of the receptor were determined by alanine scanning mutagenesis of putative binding residues. Fragment analysis of MDP revealed that both the peptide and carbohydrate portion contribute to the binding interaction. Introduction The composition of the microbiome plays a crucial role in maintaining homeostasis and when altered can increase disease susceptibility in the host 1. To properly modulate the daily flux of the microbial composition in the body, the innate immune system detects molecular patterns that trigger a corrective response to the environment. When the innate immune receptors are unable to overcome a disturbance in the microbiome, disease susceptibility increases. Crohn’s disease is an increasingly prevalent inflammatory bowel disorder2, which is proposed to occur due to an atypical response to bacteria leading to dysbiosis and uncontrolled inflammation3-5. The innate immune receptor nucleotide-binding oligomerization domaincontaining 2 (Nod2) was the first and most significant protein associated with the development of Crohn’s disease6, 7.



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Nod2 responds to a molecular signature of bacterial peptidoglycan called muramyl dipeptide (MDP)8, 9, present in both Gram-positive and Gram-negative bacteria. Nod2 directly binds to MDP10, 11, leading to an immune response through the NF-κB pathway8. The Crohn’s associated mutants of Nod2 have an altered response to MDP in comparison to the wild type (WT) 9. Recently, we have shown that the Crohn’s associated mutants are still capable of binding to MDP, albeit with slightly lower binding affinities 6, 12. The stability of the Crohn’s mutants was shown to be greatly decreased in comparison to the WT13. The signaling ability of the Crohn’s mutants was rescued through interactions with Hsp70, making the characterization of all Nod2’s binding interactions (both proteins and small molecules) critical for correcting the function of the Crohn’s mutants13, 14. The molecular basis for Nod2 recognition of MDP has long remained inconclusive. It is hypothesized that binding events occur in the leucine-rich repeat (LRR) domain15. This motif is evolutionarily conserved in many proteins associated with innate immunity in both plants and mammals16. LRRs bind to a wide range of pathogen-associated molecular patterns (PAMPs) including components of bacteria, fungi and viruses17 making it the presumptive site of MDP recognition. In mammals, both the Toll-like and Nod-like receptors (TLRs and NLRs) contain the LRR domain. Many of the TLRs such as TLR2 and TLR4 recognize bacterial components through their LRRs18, 19. In addition to Nod2, two other NLR proteins, NALP1 NLRP1 and NALP3 NLRP3, are triggered by MDP20, 21. The LRR region of NALP1 NLRP1 was crystallized but a co-crystal with MDP was unable to be obtained22. Recently, Shimizu and coworkers solved the Nod2 structure and speculated a putative carbohydrate electron density in the LRR, but could not define this as MDP23. Even with these developments, there exists limited information about LRR proteins interacting with MDP on a molecular level. An alternative


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hypothesized function of the LRR domain is to engage in protein-protein interactions16, 24. Conversely, neither of these models is supported by conclusive biochemical or biophysical data for Nod2. All studies that have sought to define the critical regions of MDP and the LRR for recognition have used cell-based assays to evaluate the effect on signaling15 23. These studies did not take into account the effect point mutations would have on the structure or stability of Nod2. An in vitro characterization of this interaction would therefore be complementary to the current cell-based assays. A limiting factor for classical biochemical characterization was the expression and purification of Nod2. Recently, the expression and purification of human Nod2 from insect cells was determined but the protein yields were too low for biochemical characterizations 10, 11. Here we report the first expression and purification of the LRR domain of human Nod2 from E. coli that leads to a high yielding, more soluble construct. Using this LRR expression construct we report conclusive evidence that the binding region for MDP is on the concave surface of the LRR region using alanine scanning mutagenesis and surface plasmon resonance (SPR). Component analysis of MDP was used to probe critical interactions of the binding pocket, and MDP was modeled into the binding pocket using the program Autodock in a conformation that was consistent with our experimental data. Results and Discussion Our previous attempts to purify full length Nod2 using E.coli were unsuccessful, likely due to its large size, membrane association and its ability to bind peptidoglycan fragments inherent in the E.coli system. A revised method to purify the LRR domain of Nod2 was pursued as a fusion with maltose-binding protein (MBP) to aid in solubility and to exclude the protein



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from inclusion bodies25. The LRR construct was designed to have the highest degree of similarity to that of ribonuclease inhibitor (RI), a LRR protein commonly used to develop homology models of Nod2 because of their high similarity26. Our homology model extended the classically defined LRR domain of Nod2 by 26 amino acids (residues 765-1040). The LRR-MBP fusion purified with roughly 90% purity and yields of 3 mg/L of expression (Figure S1). The protein was verified to have secondary structural elements by circular dichroism (CD), suggesting that the construct is folded. Moreover, we note this folding pattern was unaffected by the addition of MDP ligand (Figure S2a). The CD spectra indicate that the fusion is α-helical rich as noted by minima at 210 and 230 nm, agreeing with the secondary structural elements of the LRR domain from the crystal structure of both Nod223 and the structure of MBP27. This is the first reported purification of human Nod2 in E.coli, allowing for biochemical studies of Nod2 to be more readily accessible, which has been a limitation in the field for 15 years. The MBP-fusion construct becomes an excellent solution for studying difficult peptidoglycan-binding proteins. As the LRR was expressed bacteria, which is surrounded by peptidoglycan, special precautions were taken to avoid co-purifying fragments bound to the LRR, such as the avoidance of peptidoglycan digestive enzymes in the purification procedure, gentle lysis conditions and pH conditions that were suboptimal for binding. It has previously been shown that the binding between MDP and Nod2 is pH dependent, binding in acidic pH (

Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine

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