Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition

Abstract

Multiple genetic variants of CARD15/NOD2 have been associated with susceptibility to Crohn's disease and Blau syndrome. NOD2 recognizes muramyl dipeptide (MDP) derived from bacterial peptidoglycan (PGN), but the molecular basis of recognition remains elusive. We performed systematic mutational analysis to gain insights into the function of NOD2 and molecular mechanisms of disease susceptibility. Using an archive of 519 mutations covering approximately 50% of the amino-acid residues of NOD2, the essential regulatory domains and specific residues of NOD2 involved in recognition of MDP were identified. The analysis revealed distinct roles for N-terminal and C-terminal leucine-rich repeats (LRRs) in the modulation of NOD2 activation and bacterial recognition. Within the C-terminal LRRs, variable residues predicted to form the beta-strand/betaturn structure were found to be essential for the response to MDP. In addition, we analyzed NOD1, a NOD2-related protein, revealing conserved and nonconserved amino-acid residues involved in PGN recognition. These results provide new insights into the molecular function and regulation of NOD2 and related NOD family proteins.

Figure 1

Strategy for NOD2 mutagenesis and summary of studied NOD2 mutations. (A) Schematic representation of NOD2 including mutagenized cassettes (I–IV). The location of CARDs, NOD domain and leucine-rich repeat domain (LRD) are shown. Numbers represent the position of amino-acid residues. Restriction enzyme sites flanking each cassette are shown. (B) Summary of NOD2 mutations generated and analyzed. WT, wild-type; ND, not determined.

Figure 2

Mutational analyses of the CARD of NOD2. (A) Schematic representation of the N-terminus region of NOD2. Numbers indicate the position of amino-acid residues. Loss-of-function mutations are shown in black. Clones with wild-type activity are depicted in gray and mutations found in complex mutant clones (i.e. more than one amino-acid substitution) with wild-type activity are shown in gray italic. Mutations associated with reduced expression are labelled with the symbol ^a. Mutations found in Crohn's patients are labelled with the symbol ^b. (B) NF-κB activity of representative CARD mutants. E778K and ΔLRR are shown as controls. Values represent the mean of normalized data±s.d. of triplicate cultures. (C) Interaction of NOD2 mutants with RICK. Extracts from HEK293T cells expressing indicated mutants were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-NOD2 antibody. IP, immunoprecipitation; total, immunoblotting of total lysates. A black dot denotes the mobility shift of the phosphorylated form of RICK. (D) Inhibition of RICK-induced NF-κB activation by CARD mutants and PEA15 control plasmid. Values represent the mean of normalized data±s.d. of triplicate cultures. (E) Specific inhibition of MDP-induced NOD2-mediated NF-κB by the L145P mutant. An LPS preparation containing MDP-like activity (closed bars) was used in this assay. (F) Inhibition of MDP-induced NF-κB activation by the L145P mutant and dominant-negative form of IKKγ in HEK293T cells. DN denotes dominant-negative form.

Figure 3

Mutational analyses of the NOD domain of NOD2. (A) Schematic representation of the NOD domain. The Walker's A and B boxes are depicted by solid dots. Numbers indicate the position of amino-acid residues. Symbols and color code are described in Figure 2. The gain-of-function mutations are labelled with the symbol ^c. (B) NF-κB activity of representative NOD domain mutants in the presence or absence of MDP. E778K and ΔLRR are shown as controls. Expression of NOD2 proteins is shown on top. (C) Interaction of NOD2 mutants with RICK. Extracts from HEK293T cells expressing indicated mutants were co-immunoprecipitated with anti-Myc antibody and immunoblotted with anti-NOD2 antibody. IP, immunoprecipitation; total, immunoblotting of total lysates. A black dot denotes phosphorylated form of RICK. (D) Evidence of intramolecular interaction between the C-terminal region of the NOD domain and proximal LRRs by functional complementation. NF-κB activity in the presence or absence of MDP is shown for NOD2 mutant and wild-type clones. Values represent the mean of normalized data±s.d. of triplicate cultures.

Figure 4

Analysis of truncated NOD2 mutants. The location of CARDs, NOD domain and LRD are shown. The Walker's A and B boxes in the NOD domain are depicted by solid dots. The basal NF-κB activity (BNA) and NF-κB response to bacterial components (BR) are shown. +, response similar to that obtained with the wild-type protein; −, no response above control plasmid; +++, response at least three-fold greater than that obtained with wild-type NOD2. Representative results are shown in Figure 3. Numbers represent the position of amino-acid residues. The names of individual mutations are shown on the left. WT, wild-type clone.

Figure 5

Analyses of truncated mutants reveal an inhibitory domain in NOD1 and NOD2. (A) NOD2 activity in the absence (open bars) and presence of LPS preparation (closed bars). A schematic diagram of part of NOD and LRD of NOD2 is shown. Numbers indicate the position (amino-acid residue) of C-terminal truncations. WT, wild-type. E778K represents a control loss-of-function mutant. (−) represents results obtained with control plasmid. (B) Immunoblotting analysis of NOD2 mutants. Analysis was performed with cell extracts from HEK293T cells transfected with indicated mutant or wild-type NOD2 plasmid or control plasmid (−) and immunoblotted with anti-FLAG antibody. (C) NF-κB activity of NOD domain gain-of-function mutants in the presence or absence of MDP. Wild-type (WT) and mock cells (−) are shown as controls. Values represent the mean of normalized data±s.d. of triplicate cultures. (D) NOD1 activity in the absence (open bars) and presence of LPS preparation (closed bars). A schematic diagram of part of NOD and LRD of NOD1 is shown. Numbers indicate the position (amino-acid residue) of C-terminal truncation. WT, wild-type. (E) Immunoblotting analysis of NOD1 proteins. The analysis was performed with cell extracts from HEK293T cells transfected with indicated mutant and wild-type NOD1 plasmids or control plasmid (−) and immunoblotted with anti-NOD1 antibody.

Figure 6

Mutational analyses of the LRRs of NOD2. NF-κB activity of LRR mutants in response to MDP. Values represent the mean of normalized data±s.d. of triplicate cultures. Expression of NOD2 proteins is shown at the bottom.

Figure 7

Computer-assisted molecular modelling of the LRRs of NOD2. (A) The predicted structure of the 11 LRRs of NOD2. (B) Location of amino-acid residues mutagenized. (C) Position of loss-of-function NOD2 mutants with single amino-acid substitutions in the LRRs. Mutations located in α-helix/turn regions and β-strands are depicted in white and yellow, respectively. The E778K, E843K and G908R mutants (underlined) have been found in CD patients. (D) Amino-acid alignment of the turn region and β-strands of LRRs of NOD2. Letters denote amino-acid residue. X denotes any amino acid. The locations of residues involved in amino-acid substitutions with wild-type and loss-of-function phenotype are shown in green and yellow, respectively. The specific amino-acid replacements are shown in Figure 6 and Supplementary Table 1.