Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

Synthesis of Conformationally Constrained d-Glu-meso-DAP Analogs as Innate Immune Agonists.

The dipeptide d-Glu-meso-DAP (iE-DAP) is the minimal structural fragment capable of activating the innate immune receptor nucleotide-binding oligomerization domain protein (NOD1). The meso-diaminopimelic acid (meso-DAP) moiety is known to be very stringent in terms of the allowed structural modifications which still retain the NOD1 activity. The aim of our study was to further explore the chemical space around the meso-DAP portion and provide a deeper understanding of the structural features required for NOD1 agonism. In order to achieve the rigidization of the terminal amine functionality of meso-DAP, isoxazoline and pyridine heterocycles were introduced into its side-chain. Further, we incorporated the obtained meso-DAP mimetics into the structure of iE-DAP. Collectively, nine innovative iE-DAP derivatives additionally equipped with lauroyl or didodecyl moieties at the α-amino group of d-Glu have been prepared and examined for their NOD1 activating capacity. Overall, the results obtained indicate that constraining the terminal amino group of meso-DAP abrogates the compounds' ability to activate NOD1, since only compound 6b retained noteworthy NOD1 agonistic activity, and underpin the stringent nature of this amino acid with regard to the allowed structural modifications.

Harnessing the untapped potential of nucleotide-binding oligomerization domain ligands for cancer immunotherapy.

In the last decade, cancer immunotherapy has emerged as an effective alternative to traditional therapies such as chemotherapy and radiation. In contrast to the latter, cancer immunotherapy has the potential to distinguish between cancer and healthy cells, and thus to avoid severe and intolerable side-effects, since the cancer cells are effectively eliminated by stimulated immune cells. The cytosolic nucleotide-binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are important components of the innate immune system and constitute interesting targets in terms of strengthening the immune response against cancer cells. Many NOD ligands have been synthesized, in particular NOD2 agonists that exhibit favorable immunostimulatory and anticancer activity. Among them, mifamurtide has already been approved in Europe by the European Medicine Agency for treating patients with osteosarcoma in combination with chemotherapy after complete surgical removal of the primary tumor. This review is focused on NOD receptors as promising targets in cancer immunotherapy as well as summarizing current knowledge of the various NOD ligands exhibiting antitumor and even antimetastatic activity in vitro and in vivo.

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective.

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in Asian seabass, Lates calcarifer: Cloning, ontogeny and expression analysis following bacterial infection or ligand stimulation.

NOD1 (Nucleotide-binding oligomerization domain-containing protein 1) is one of the most prominent intracellular Nod-like receptors (NLRs), responsible for detecting different microbial components and products arising from tissue injury. Here, we have identified and cloned NOD1 transcript in the Asian seabass, Lates calcarifer (AsNOD1), which consists of 3749 nucleotides and encodes for a predicted putative protein of 900 AA. The AsNOD1 possesses the typical structure of NLR family, consisting of N-terminal CARD domain, centrally located NACHT domain and C-terminal LRRs. The AsNOD1 showed ubiquitous tissue expression in 11 different tissues of healthy animals tested with high levels of expression in hindgut and gill. From the ontogenetic expression profile of AsNOD1, it is quite evident that this gene might follow a maternally-transferred trend in euryhaline teleosts, as it is highly abundant in embryonic developmental stages. The constitutive immunomodulation of AsNOD1 in terms of expression level was clearly evident in the different tissues of Asian seabass-injected either with Vibrio alginolyticus or poly I:C. However, injection with Staphylococcus aureus did not elicit similar immunomodulation except for the up-regulation noticed at few time-points in some tissues. SISK-cell line induced with different ligands such as poly I:C, LPS and PGN also showed up-regulation of AsNOD1 in certain time-points in vitro. Based on the results obtained in the present study, it can be inferred that the AsNOD1 might play an immunoregulatory role upon exposure to different bacterial as well as viral PAMPs and also might be an important component of innate immune element during embryonic and larval development in the euryhaline teleost Asian seabass.

Understanding the molecular differential recognition of muramyl peptide ligands by LRR domains of human NOD receptors.

Human nucleotide-binding oligomerization domain proteins, hNOD1 and hNOD2, are host intracellular receptors with C-terminal leucine-rich repeat (LRR) domains, which recognize specific bacterial peptidoglycan (PG) fragments as their ligands. The specificity of this recognition is dependent on the third amino acid of the stem peptide of the PG ligand, which is usually meso-diaminopimelic acid (mesoDAP) or l-lysine (l-Lys). Since the LRR domains of hNOD receptors had been experimentally shown to confer the PG ligand-sensing specificity, we developed three-dimensional structures of hNOD1-LRR and the hNOD2-LRR to understand the mechanism of differential recognition of muramyl peptide ligands by hNOD receptors. The hNOD1-LRR and hNOD2-LRR receptor models exhibited right-handed curved solenoid shape. The hot-spot residues experimentally proved to be critical for ligand recognition were located in the concavity of the NOD-LRR and formed the recognition site. Our molecular docking analyses and molecular electrostatic potential mapping studies explain the activation of hNOD-LRRs, in response to effective molecular interactions of PG ligands at the recognition site; and conversely, the inability of certain PG ligands to activate hNOD-LRRs, by deviations from the recognition site. Based on molecular docking studies using PG ligands, we propose few residues - G825, D826 and N850 in hNOD1-LRR and L904, G905, W931, L932 and S933 in hNOD2-LRR, evolutionarily conserved across different host species, which may play a major role in ligand recognition. Thus, our integrated experimental and computational approach elucidates the molecular basis underlying the differential recognition of PG ligands by hNOD receptors.

Bacterial peptidoglycan with amidated meso-diaminopimelic acid evades NOD1 recognition: an insight into NOD1 structure-recognition.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an intracellular pattern recognition receptor that recognizes bacterial peptidoglycan (PG) containing meso-diaminopimelic acid (mesoDAP) and activates the innate immune system. Interestingly, a few pathogenic and commensal bacteria modify their PG stem peptide by amidation of mesoDAP (mesoDAPNH2). In the present study, NOD1 stimulation assays were performed using bacterial PG containing mesoDAP (PGDAP) and mesoDAPNH2 (PGDAPNH2) to understand the differences in their biomolecular recognition mechanism. PGDAP was effectively recognized, whereas PGDAPNH2 showed reduced recognition by the NOD1 receptor. Restimulation of the NOD1 receptor, which was initially stimulated with PGDAP using PGDAPNH2, did not show any further NOD1 activation levels than with PGDAP alone. But the NOD1 receptor initially stimulated with PGDAPNH2 responded effectively to restimulation with PGDAP The biomolecular structure-recognition relationship of the ligand-sensing leucine-rich repeat (LRR) domain of human NOD1 (NOD1-LRR) with PGDAP and PGDAPNH2 was studied by different computational techniques to further understand the molecular basis of our experimental observations. The d-Glu-mesoDAP motif of GMTPDAP, which is the minimum essential motif for NOD1 activation, was found involved in specific interactions at the recognition site, but the interactions of the corresponding d-Glu-mesoDAP motif of PGDAPNH2 occur away from the recognition site of the NOD1 receptor. Hot-spot residues identified for effective PG recognition by NOD1-LRR include W820, G821, D826 and N850, which are evolutionarily conserved across different host species. These integrated results thus successfully provided the atomic level and biochemical insights on how PGs containing mesoDAPNH2 evade NOD1-LRR receptor recognition.

Molecular cloning and functional analysis of nucleotide-binding oligomerization domain-containing protein 1 in rainbow trout, Oncorhynchus mykiss.

NOD1 has important roles in innate immunity as sensor of microbial components derived from bacterial peptidoglycan. In this study, we identified genes encoding components of the NOD1 signaling pathway, including NOD1 (OmNOD1) and RIP2 (OmRIP2) from rainbow trout, Oncorhynchus mykiss, and investigated whether OmNOD1 has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 treated with NOD1-specific ligand (iE-DAP). The deduced amino acid sequence of OmNOD1 contained conserved CARD, NOD and LRR domains. Loss-of-function and gain-of-function experiments indicated that OmNOD1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmNOD1 in RTH-149 cells treated with iE-DAP decreased the expression of IL-1ß, IL-6, IL-8 and TNF-α. Conversely, overexpression of OmNOD1 resulted in up-regulation of IL-1ß, IL-6, IL-8 and TNF-α expression. In addition, RIP2 inhibitor (gefitinib) significantly decreased the expression of these pro-inflammatory cytokines induced by iE-DAP in RTH-149 cells. These findings highlight the important role of NOD1 signaling pathway in fish in eliciting innate immune response.

Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2.

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1.

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 µM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 µM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 µM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.

Molecular cloning and characterization of nucleotide binding and oligomerization domain-1 (NOD1) receptor in the Indian Major Carp, rohu (Labeo rohita), and analysis of its inductive expression and down-stream signalling molecules following ligands exposure and Gram-negative bacterial infections.

Nucleotide binding and oligomerization domain-1 (NOD1) is a cytoplasmic pattern recognition receptor (PRR), and is a member of the NOD-like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products, and plays a key role in inducing innate immunity. In this report, NOD1 gene was cloned and characterized in rohu (Labeo rohita), a fish species of highest commercial importance in the Indian subcontinent. The full-length rohu NOD1 (rNOD1) cDNA comprised of 3168 bp with a single open reading frame (ORF) of 2814 bp, encoding a polypeptide of 937 amino acids (aa) with an estimated molecular mass of 106.13 kDa. Structurally, it comprised of one caspase recruitment domain (CARD) at N-terminal, seven leucine rich repeat (LRR) regions at C-terminal and one NACHT domain in between N and C-terminals. Phylogenetically, rNOD1 was closely related to grass carp NOD1 (gcNOD1), and exhibited significant similarity (95.8%) and identity (91.0%) in their amino acids. Ontogenic expression analysis of rNOD1 and its associated down-stream signaling molecule RICK (receptor interacting serine­threonine kinase) by quantitative real-time PCR (qRT-PCR) revealed their constitutive expression in all embryonic developmental stages. Basal expression analysis of rNOD1 showed its wide range of expression in all examined tissues, highest was in spleen and the lowest was in blood. Inductive expression of rNOD1 was observed following LPS and poly I:C exposure, and Aeromonas hydrophila, Edwardsiella tarda and Shigella flexneri infections. Expression of RICK in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with the inductive expression of rNOD1. Together, these findings highlighted the important role of NOD1 in fish in response to pathogenic invasion.

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

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 ß-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.

NOD-like receptors (NLRs): bona fide intracellular microbial sensors.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) (nucleotide-binding domain leucine-rich repeat containing) family of proteins has been demonstrated to function as regulators of innate immune response against microbial pathogens. Stimulation of NOD1 and NOD2, two prototypic NLRs, results in the activation of MAPK and NF-kappaB. On the other hand, a different set of NLRs induces caspase-1 activation through the assembly of an inflammasome. This review discusses recent findings regarding the signaling pathways utilized by NLR proteins in the control of caspase-1 and NF-kappaB activation, as well as the nonredundant role of NLRs in pathogen clearance. The review also covers advances regarding the cellular localization of these proteins and the implications this may have on pathogen sensing and signal transduction.

Identification of benzofused five-membered sultams, potent dual NOD1/NOD2 antagonists in vitro and in vivo.

Nucleotide-binding oligomerization domain-containing proteins 1 and 2 play important roles in immune system activation. Recently, a shift has occurred due to the emerging knowledge that preventing nucleotide-binding oligomerization domains (NODs) signaling could facilitate the treatment of some cancers, which warrants the search for dual antagonists of NOD1 and NOD2. Herein, we undertook the synthesis and identification of a new class of derivatives of dual NOD1/NOD2 antagonists with novel benzofused five-membered sultams. Compound 14k was finally demonstrated to be the most potent molecule that inhibits both NOD1-and NOD2-stimulated NF-κB and MAPK signaling in vitro and in vivo.

The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction.

The pattern-recognition molecule Nod1 is a critical sensor for bacterial derived diaminopimelic acid-containing peptidoglycan fragments which induces innate immune responses in epithelial cells. Here we report the subcellular localization of this protein in human epithelial cells. Nod1 is localized in the cytosol and at the plasma membrane in human cells. This membrane association is dependent on the integrity of the protein, on its signalling capacity and on an intact actin cytoskeleton. Signalling-inactive mutants of Nod1 or disruption of the actin cytoskeleton interferes with this localization pattern and impacts on downstream NF-kappaB activation. Moreover, the invasive bacterium Shigella flexneri was used as a model for physiological activation of Nod1. Imaging revealed that Nod1 is recruited to the site of bacterial entry, where it colocalizes with NEMO. Our data provide evidence that membrane association is linked to Nod1 function and, in view of recent findings on Nod2, that this may be a common feature of NLR family members.

Sensing intracellular pathogens-NOD-like receptors.

The innate immune system uses different molecules that sense pathogen associated molecular patterns. These include Toll-like receptors (TLRs), RIG-1-like receptors (RLRs) and the NOD-like receptors (NLRs). The NLRs, consisting of more than 20 related family members, are present in the cytosol and recognize intracellular ligands. Members of the NLR can be grouped into molecules that contain either a CARD or a Pyrin motif. The NOD proteins mediate NF-kappaB activation, whereas Pyrin molecules such as NALP3 regulate IL-1beta and IL-18 production. In this review, we will discuss the role of NLRs in pattern recognition of microbial components and their role in health and disease.

Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.

The nucleotide oligomerization domain (NOD)-like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling.

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.

Solution structure of NOD1 CARD and mutational analysis of its interaction with the CARD of downstream kinase RICK.

NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.

CARD domain of rat RIP2 kinase: Refolding, solution structure, pH-dependent behavior and protein-protein interactions.

RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.