The Nod-Like Receptor (NLR) Family: A Tale of Similarities and Differences

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs posses a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs

Evaluation of Nod-Like Receptor (NLR) Effector Domain Interactions

Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-κB activation

Strength of selected interactions in terms of HIS3 reporter gene activation.

<p>The maximum concentration of 3-aminotrizol supporting visible growth of transformants is indicated.</p

NLR protein-protein interactions.

<p>Respective protein-protein interactions were mined from the scientific literature or retrieved from MiMI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Jayapandian1" target="_blank">[65]</a>. Results represent data from our yeast two-hybrid analysis (interaction observed or not observed). Baits and preys indicate individual domains tested in our analysis with interacting constructs in bold. References to reported interactions are specified.</p>§<p>Initially not observed by “each against all” approach, but observed subsequently with refined constructs.</p>*<p>No data on a direct interaction.</p

RIPK2 CARD forms homodimers/-oligomers.

<p><i>Left panel</i>, Yeast two-hybrid analysis revealed that RIPK2 CARD (residues 427–527) forms homodimers/-oligomers (column 1 on SD/-4). Lamin c (Lam c) was used as control (column 5). <i>Right panel</i>, GST pull down assay. Specific binding of ³⁵S-labeled RIPK2 CARD (residues 427–527) was observed to recombinant expressed GST-RIPK2 CARD (residues 427–527), whereas binding to GST-Lam c was not detected. SD/-2: SD/-Leu/-Trp, SD/-4: SD/-Ade/-His/-Leu/-Trp.</p

Cloning of constructs.

<p>GenBank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Benson1" target="_blank">[66]</a> accession numbers, featured domains as well as corresponding amino acid residues are indicated, respectively.</p>*<p>NOD2 tripled mutation – E69K, D70K, D71K; CARD: caspase activation and recruitment domain, PYD: pyrin domain.</p

The interaction matrix.

<p>In an “each against all” approach an overall number of 676 (26×26) effector domain combinations were analyzed. A “+” indicates an interaction between a particular pair, whereas “−” symbolizes no interaction. In total, the approach yielded 25 distinct associations, which actually corresponded to 7 unique pairs of interacting proteins, as well as 5 homodimerizations.</p

NOD2 interaction with distinct NLRP members.

<p><i>Upper panel</i>, A “+” indicates an interaction, “−” symbolizes no interaction (n.d.: not done). A short isoform of NOD2 (NOD2-S, residues 1–180), maintains the interaction with NLRP1, -3, and -12. Furthermore, a linker region within NLRP1 (residues 92–341) proved sufficient for interaction with NOD2. <i>Lower panel</i>, Schematic illustration of particular NOD2/NLRP1 constructs and their respective interactions. FIIND: Function to find domain.</p

NOD2 directly interacts with NLRP1, NLRP3, and NLRP12.

<p><i>Left panel</i>, Yeast two-hybrid analysis of NOD2 CARDs (residues 1–267) showed interaction with NLRP1,-3 and -12, but not with other NLRP proteins (NLRP2, -7, -10, or -11; column 1). Lam c was used as a negative control (column 2). Interestingly, NOD2 mutant E69K maintained the binding to NLRP1, -3, and -12 (column 3), whereas NOD2 D70K (column 4) as well as the NOD2 triple mutant (3xmut, column 5) did not. An unrelated mutation not located within the prospective interaction interface had no effect (column 6). <i>Right panel</i>, Physical interaction of NOD2 and NLRP3 in human cells. Western analysis of lysates (IN) and immunoprecipitated complexes (IP) from HEK293T cells, transiently transfected with expression plasmid encoding human HA-NOD2 and FLAG-NLRP3. NLRP3 was immunoprecipitated from cell lysates using a FLAG-epitope specific antibody. Proteins were detected using anti-HA and anti-FLAG antibodies, respectively. As negative control, proteins were immunoprecipitated with FLAG-epitope specific antibody from lysates of HEK293T cells transiently transfected with HA-NOD2, but not FLAG-NLRP3.</p