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

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

Interaction of distinct NOD2 mutants with RIPK2.

<p><i>Left panel</i>, Yeast two-hybrid analysis. NOD2 CARD1+2 interacts with RIPK2 CARD (column 1). No interactions were observed for NOD2 variants harboring a disrupted acidic patch (transformation 2, 3, and 4). An unrelated mutation not affecting the interaction interface had no effect (column 5). <i>Right panel</i>, Autoradiography of an <i>in vitro</i> transcription/translation of NOD2 proteins. NOD2 constructs were expressed using similar amounts of DNA and lysates.</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

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

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

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

Multiple sequence alignment of NLR NACHT-WH-SH domains and the Apaf-1 NACHT-WH-SH domain.

<p>Degree of conservation is shown as blue shading. The secondary structure of Apaf-1 is shown above the Apaf-1 sequence. Arrows underneath the alignment indicate domain boundaries. Conserved sequence features important for catalytic activity are shown in black boxes. Orange and magenta boxes depict interfaces residues while the orange ones contribute to interactions with the left partner and the magenta residues are thought to interact with the right partner in the oligomer. Green boxes indicate additional motifs as described in the text.</p

Figure 5

<p>A Multiple sequence alignment of NLR and Apaf-1 CARD domains. Acidic key residues participating in the CARD-CARD interface are indicated by red borders. Residues that belong to the basic patch of the CARD-CARD interface are indicated by blue borders. Nod2.1 and Nod2.2 refer to NOD2 CARD domain 1 and 2, respectively. B Multiple sequence alignment of NLR PYRIN domains. Patch of negatively charged residues from ASC2 in helices 1 and 4 and their corresponding residues in the PYRIN domain containing NLR proteins (red box). Patch of positively charged residues from ASC2 in helices 2 and 3 and their corresponding residues in the PYRIN containing NLR proteins (blue box).</p