Fold and stability of the CARDs of NOD2.

<p>(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.</p

Characterisation of the interaction between the tandem SH3 domains of NOXO1 and peptides p22^phox.

<p>Upper part shows the raw calorimetric data for the interaction of p22^phoxC and human NOXO1 SH3_AB. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) Human p22^phoxC titrated into human NOXO1 SH3_AB (▴) Mouse p22^phoxC titrated into mouse NOXO1 SH3_AB.</p

The interaction of RIP2 CARD mutants with NOD2 CARDab and NOD1 CARD.

<p>Presence (+)/absence (−) of RIP2 CARD on the beads following coexpression and GST-pull downs as analyzed by SDS-PAGE.</p

Characterisation of intermolecular interactions between NOXO1 and NOXA1.

<p>All measurements were performed at 18°C. The <i>K</i>_d is given in units of 10⁻⁶ M, Δ<i>H</i> and TΔ<i>S</i> are given in kcal mol⁻¹ (1 kcal/mol≡4.184 kJ/mol). The stoichiometry of complex formation for each binding site is N = 1.0±0.1.</p

Schematic representation of the domain structures of p47^phox, NOXO1, p67^phox and NOXA1.

<p>(<b>A</b>) Predicted domain structures of NOXO1 and NOXA1 in comparison to p47^phox and p67^phox, respectively. Human and mouse constructs used in this study are illustrated by black lines. Mouse constructs are identical in length unless otherwise stated in brackets. The autoinhibitory region (AIR) and proline rich region (PPR) are indicated. (<b>B</b>) Alignment of p47^phox, human NOXO1 and mouse NOXO1. The PX domains (grey shaded), SH3 domains (cyan shaded), polybasic region (orange shaded ox) and proline rich motif (green shaded) are indicated. (<b>C</b>) Structure of the autoinhibited core of p47^phox showing the superSH3 domain conformation. The structure shows the biologically relevant monomeric form of the protein that is also observed in solution, not the domain-swapped crystallized dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010478#pone.0010478-Groemping2" target="_blank">[22]</a>. The SH3 domains and polybasic region are highlighted in cyan and orange, respectively.</p

Characterisation of intermolecular interactions between NOXO1 and p22^phox.

<p>All measurements were performed at 18°C. The <i>K</i>_d is given in units of 10⁻⁶ M, Δ<i>H</i> and TΔ<i>S</i> are given in kcal mol⁻¹ (1 kcal/mol≡4.184 kJ/mol). The stoichiometry of complex formation for each binding site is N = 1.0±0.1. No binding is indicated by NB.</p

Residues involved in the NOD2-RIP2 interaction.

<p>(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.</p

Characterisation of the interaction between NOXO1 and NOXA1.

<p>(<b>A</b>) Upper part shows the raw calorimetric data for the interaction between human NOXA1 SH3 and NOXO1 SH3_AB–E. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) Human NOXA1 SH3 titrated into human NOXO1 SH3_AB–E (▴) Mouse NOXA1 SH3 titrated into mouse NOXO1 SH3_AB–E. (<b>B</b>) Upper part shows raw calorimetric data for the interaction of peptideA and human NOXA1 SH3. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) PeptideA titrated into human NOXA1 SH3 (▴) PeptideA titrated into mouse NOXA1 SH3.</p

Characterisation of the interaction between NOXO1 SH3_AB–E and p22^phox.

<p>Upper part shows the raw calorimetric data for the interaction of mouse p22^phoxC- NOXO1 SH3_AB–E. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) Human p22^phoxC titrated into human NOXO1 SH3_AB–E (▴) Mouse p22^phoxC titrated into mouse NOXO1 SH3_AB–E.</p

Thermodynamics of the NOD2 CARDa-CARDb interaction.

<p>(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 K_d of 1.1 µM. A control experiment of CARDa into buffer is shown. (B) Determination of the heat capacity, ΔC_p. Enthalpies, ΔH, from CARDa-CARDb titrations at different temperatures were plotted against the temperatures. Linear regression analysis gave ΔC_p = 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).</p