Figure 1.
Oligomeric state of the CARDs of NOD2.
(A) Schematic representation of the domain structure of human NOD2 and the domain boundaries of the constructs (tandem CARD, CARDa and CARDb) used in this study. (B) AUC Sedimenation Equilibrium resulted in MW of 22 kDa±0.5 kDa for CARDab (calculated MW = 22043 Da). The sample was run at three different concentrations and three different speeds (18, 22, 26 kprm). 11 scans were collected over 4 days. (C) AUC Sedimentation Velocity resulted in MW of 10–11 kDa for CARDa (blue line, calculated MW = 11155 kDa) and CARDb (red line, calculated MW = 11403 kDa), respectively. Samples were run at OD600∼0.5. SedFit was used for data analysis. A sample containing equal molar amounts of CARDa and CARDb displayed a MW of ∼18 kDa.
Figure 2.
Fold and stability of the CARDs of NOD2.
(A) Far-UV CD at 250–195 nm showed highly α-helical proteins as reflected in the double minima at 208 and 222 nm and the strong positive band at 195 nm. Sample concentration was 0.15 mg/ml. The black curve shows the mean residue ellipticity of NOD2-CARDab, the blue curve of NOD2-CARDa and the red curve of NOD2-CARDb. (B) CD thermal unfolding from 5 to 95°C at 222 nm. Sample concentration was 0.15 mg/ml. A 2 mm cuvette was used. The black curve represents the mean residue ellipticity of NOD2-CARDab, the blue curve of CARDa and the red curve of CARDb. The mixture contained 0.075 mg/ml of NOD2-CARDa and NOD2-CARDb, respectively, and is shown in green. The grey curve represents the computed mean value of NOD2-CARDa and NOD2-CARDb.
Figure 3.
Thermodynamics of the NOD2 CARDa-CARDb interaction.
(A) ITC measurement of complex formation between CARDa in the syringe (475 µM) and CARDb in the cell (45 µM). T = 25°C. The binding isotherm was fitted to a one-site binding model with a Kd of 1.1 µM. A control experiment of CARDa into buffer is shown. (B) Determination of the heat capacity, ΔCp. Enthalpies, ΔH, from CARDa-CARDb titrations at different temperatures were plotted against the temperatures. Linear regression analysis gave ΔCp = dΔH/dT = −450 cal/(mole °C). (C) Effect of CARDa point mutations as monitored by ITC at 25°C. Titration of CARDa E69K (345 µM) into CARDb (40 µM) is shown in blue, CARDa E72K (205 µM) into CARDb (26 µM) in green and CARDa R86A (504 µM) into CARDb (62 µM) in red. The titrations were performed in the same buffer as in (A).
Table 1.
Thermodynamic parameters for the CARDa-CARDb interaction.
Table 2.
Salt dependence of the CARDa-CARDb interaction.
Figure 4.
Complex formation between NOD2 CARDab and RIP2-CARD.
(A) NOD2 CARDab with a N-terminal GST-tag and RIP2-CARD equipped with a N-terminal GB1-tag and a C-terminal His6-tag were co-expressed and pulled-down with glutathione sepharose beads. From left: Lane 1) Protein Marker, GE Healthcare. Lane 2) Soluble lysate. Lane 3) Bead eluate after 3C-protease cleavage. NOD2-CARDab 22.0 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. Lane 4) Supernatant bound to beads. GST-NOD2 CARDab 48.5 kDa (green) and GB1-RIP2 CARD-His 18.6 kDa (red) are indicated by arrows. (B) Effect of NOD2 CARDab single point mutations on RIP2 CARD binding. A representative cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. * = residual expression of GST. (C) Effect of RIP2 CARD single point mutations on NOD2 CARDab binding. A cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. (D) Effect of RIP2 CARD single point mutations on NOD1 CARD binding. A cross section of the mutants tested are shown. GST-NOD1 CARD 40.9 kDa (aa17–138) and GB1-RIP2 CARD-His 18.6 kDa are indicated by blue and red arrows, respectively.
Figure 5.
Residues involved in the NOD2-RIP2 interaction.
(A) Structure of the CARD-CARD complex between Apaf-1 (light blue) and procaspase-9 (yellow), pdb ID 3YGS. The relative location of residues that were identified to disrupt the NOD2-RIP2 interaction has been mapped onto the CARD-CARD structure, based on the alignment shown in (B). These include R38 and R86 (shown in dark blue) located in CARDa of NOD2 that are shown mapped onto the CARD of procaspase-9 and D461, E472, D473, E475 and D492 in RIP2 (shown in red), mapped onto the CARD of Apaf-1. (B) The featured residues are highly conserved in CARDs. CARDa R38 and R86 correspond to two (R13 and R56) of the three basic residues in procaspase-9 (shown in blue) that are crucial for the interaction with Apaf-1. CARDa has no equivalent to the third residue, R52. Conversely, RIP2 CARD D461 corresponds to Apaf-1 D27 and RIP2 E472, D473 and E475 are located in the region of Apaf-1 E40. Apaf-1 D27 and E40 (shown in red) are both crucial for the interaction with caspase-9.
Table 3.
The interaction of NOD2 CARDab mutants with the CARD of RIP2.
Table 4.
The interaction of RIP2 CARD mutants with NOD2 CARDab and NOD1 CARD.