Crystal structure of NOD2 and its implications in human disease

Abstract

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a member of the NOD-like receptors family, are crucial for innate immune responses. Mutations of NOD2 have been associated with chronic inflammatory disorders such as Crohn's disease (CD), Blau syndrome (BS) and early-onset sarcoidosis (EOS), but little is known about its signalling mechanism and the role it plays in these diseases. Here, we report the crystal structure of rabbit NOD2 in an ADP-bound state. The structure reveals an inactive closed conformation in which the subdomains in the NOD domain are closely packed by ADP-mediated and inter-domain interactions. Mapping of the BS- or EOS-associated gain-of-function mutations reveals that most of these mutations are located in the NOD subdomain interfaces, and are likely to disrupt the inner domain interactions, facilitating a conformational change to the active form. Conversely, mutations associated with CD are distributed throughout the protein, some of which may affect oligomer formation and ligand binding.

Figure 1. Structure of OcNOD2.

(a) Structure of OcNOD2ΔCARDΔloop (Form 2). The domain organization of OcNOD2 is shown at the top. Front view (left) and side views rotated by 90° along a vertical axis relative to the left (right). OcNOD2 is comprised of five sequential domains: NBD (195–425, pink), HD1 (426–485, purple), WHD (486–602, cyan), HD2 (603–743, light green) and LRR (744–1,020, yellow). The secondary structures are labelled. The ADP molecule is shown as a stick structure with the C, O, N and P atoms coloured green, red, blue and magenta, respectively. The regions missing in the refined model are indicated by dashed lines. The colouring scheme for each domain is used throughout all of the figures. (b) The structure of mouse NLRC4 (PDB code 4KXF) displayed in nearly the same orientation as OcNOD2 ((a) left). (c) Detailed view of the ADP-binding site of OcNOD2. Residues involved in the recognition of ADP are shown as a stick structure and labelled. The 2Fo−Fc electron-density map is shown in brown mesh around ADP. Hydrogen bonds are indicated by dashed lines. Red spheres represent water molecules.

Figure 2. Inter-domain interactions in the NOD domain.

(a) Inter-domain interactions in the NOD domain viewed from two different orientations. The WHD–HD2 and HD1–WHD interfaces are highlighted by semi-transparent beige ovals. Structural elements involved in the interactions are labelled. (b–e) Magnified views of the NBD–HD2 (b), NBD–WHD (c), LRR–HD1 (d) and LRR-HD2 (e) interfaces. Each panel corresponds to the region indicated in a. Hydrogen bonds and salt bridges are shown as dashed grey lines. (f) Constitutive NF-κB activation by human NOD2 mutants associated with auto-inhibition by luciferase reporter assay using HEK293T cells. Residues in parenthesis are derived from OcNOD2. Data represent the mean relative light units (RLU) of NF-κB activity in the absence of MDP (n=6, ±s.d.). Dotted lines indicate activity of wild-type NOD2.

Figure 3. Potential ligand-binding site on the concave surface of LRR.

(a) Semi-transparent surface representation showing the concave face of the LRR domain. The potential ligand-binding site is indicated by a rectangle (left) and is magnified on the right side of the panel. The residues forming the pocket are shown as stick structures and labelled. Positive and negative electrostatic potentials are shown in blue and red, respectively. The Fo−Fc difference electron density in the pocket is contoured at the 3.0 sigma level with magenta mesh. (b) The residues in the ligand binding implicated by mutational study (left) and sequence conservation analysis (middle) are shown as blue and coral stick structures, respectively. The residues conserved among all species of NOD2 (listed in Supplementary Fig. 1) are shown in the middle panel, except for those at positions 1, 4, 6, 9 and 11, of the first 11 residues of each LRR (shown in the right panel). The residues shown in the left and middle panels are highlighted in the right panel in blue and coral, respectively. (c) MDP induced NF-κB activation of the human NOD2 mutants for the residues forming the proposed MDP-binding pocket and its peripheral region. Residues in parenthesis are derived from rabbit NOD2. Luciferase activity was measured by NF-κB-dependent luciferase reporter assay using HEK293T cells co-expressing rabbit NOD2 and human SLC15A3. Data represent the mean fold induction of NF-κB activity (n=3, ±s.d.), calculated as the relative light units (RLU) of cells stimulated with MDP divided by the RLU of non-stimulated cells. Dotted lines indicate activity of wild-type NOD2.

Figure 4. Orientation of the LRR domain.

(a–c) Superpositions of OcNOD2ΔCARD and inactive mNLRC4ΔCARD (a), OcNOD2ΔCARD and NLRC4/NLRC4 lateral dimers of NLRC4–NAIP2 inflammasome (b), and inactive mNLRC4ΔCARD and NLRC4/NLRC4 lateral dimers of NLRC4–NAIP2 inflammasome (c) using the NBD–HD1 regions for alignment (residues 195–485 and 95–355 for NOD2 and NLRC4, respectively). OcNOD2ΔCARD, inactive mNLRC4ΔCARD and NLRC4 lateral dimers of NLRC4–NAIP2 inflammasome are shown in red/pink, light blue and grey, respectively.

Figure 5. Disease-related mutations.

(a) Mapping of the BS/EOS-associated mutations in the structure of OcNOD2. Mutated residues are shown as coral spheres. (b) Detailed view of a. Mutated residues are shown as coral sticks. (c) Mapping of CD-associated mutations in the structure of OcNOD2 rotated 180° from the orientation shown in a. Mutated residues are shown as green spheres. (d) Putative oligomer model of NOD2. The oligomer model shown in cartoon (left). Magnified view of the NBD–HD1 region in the three adjacent protomers of NOD2 (right). The oligomer model is constructed by superimposing the OcNOD2 structure onto the CED4 octamers (PDB code 3LQQ) using the NBD domain as reference. The middle protomer is coloured in pink (NBD) and purple (HD1) and the rest of the protomers are shown in light colours. CD-related mutations in the NBD domain are shown as green spheres in the middle protomer and as palegreen spheres in the adjacent protomers. (e) Electrostatic surface potential of OcNOD2 shown in the same orientation as in c. CD-related mutations located on the positively charged surface of HD2 are labelled.