Molecular mechanism for NLRP6 inflammasome assembly and activation

Abstract

Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation. Here we present cryo-EM and crystal structures of NLRP6 pyrin domain (PYD). We show that NLRP6 PYD alone is able to self-assemble into filamentous structures accompanied by large conformational changes and can recruit the ASC adaptor using PYD-PYD interactions. Using molecular dynamics simulations, we identify the surface that the NLRP6 PYD filament uses to recruit ASC PYD. We further find that full-length NLRP6 assembles in a concentration-dependent manner into wider filaments with a PYD core surrounded by the NBD and the LRR domain. These findings provide a structural understanding of inflammasome assembly by NLRP6 and other members of the NLR family.

Keywords: NLRP6; X-ray crystallography; cryo-EM; inflammasome; innate immunity.

Fig. 1.

NLRP6 promotes assembly of the ASC PYD filament. (A) A cartoon framework of PYD-containing inflammasome assemblies. (B–D) FP assays of ASC^PYD filament formation nucleated by NLRP6^FL (B), NLRP6^PYD+NBD (C), and NLRP6^PYD (D). Fitted apparent dissociation constants (K_app) and Hill coefficients (n) are shown.

Fig. 2.

Cryo-EM structure of NLRP6^PYD filament. (A) A cryo-EM image of NLRP6^PYD filaments. (B) Side view of the reconstruction superimposed with three NLRP6^PYD subunit models. One subunit is enlarged for a closer view. (C) Surface representation of NLRP6^PYD filament structure, top view (Left) and side view (Right). The three main colors of red, yellow, and blue denote the threefold related helical chains. The two shades of each color are used to distinguish neighboring subunits. (D) Cartoon representation of a single NLRP6 PYD domain with six α-helices. (E) Schematic diagram of NLRP6^PYD filament, with three adjacent subunits labeled in cyan, green, and magenta. (F) Detailed interactions of type I, II, and III interfaces in the NLRP6^PYD filament.

Fig. 3.

Structure validation by mutagenesis. (A) Void and monomer fractions in the gel filtration profile of WT and mutant NLRP6^PYD-mCherry proteins. (B) FP assay of ASC^PYD filament formation nucleated by void fractions of WT and mutant NLRP6^PYD. (C) Plotted initial polymerization rates of ASC^PYD from data in B. (D) WT and mutant NLRP6^PYD-mCherry overexpressed in HeLa cells examined by confocal microscopy.

Fig. 4.

Crystal structure of NLRP6 PYD and structural comparison. (A) Overall crystal structure of NLRP6^PYD with rigidly linked MBP. (B) Structural comparison of the filament form of an NLRP6^PYD subunit (cyan) and the monomeric crystal structure of NLRP6^PYD (magenta). Shown is an enlarged view of conformational changes induced by filament formation at the α2-α3 loop. (C) Sequence alignment of the PYDs of NLRP6, AIM2, ASC, and NLRP3. The PYD filament forms of NLRP6, AIM2, and ASC are known, and the different interfacial residues in these filaments are labeled with the indicated colors. (D) Structural comparison of monomeric ASC^PYD (PDB ID code 1UCP) and monomeric NLRP6^PYD in the crystal structure. (E) Structural comparison of a single subunit of ASC^PYD filament (PDB ID code 3J63) and the NLRP6^PYD filament form. (F) Structural comparison of the AIM2^PYD monomer form (PDB ID code 3VD8) and the NLRP6^PYD filament form. (G) Structural comparison of the NLRP3^PYD crystal structure (PDB ID code 3QF2) and the NLRP6^PYD filament form.

Fig. 5.

NLRP6 PYD recruits ASC PYD with directional preference. (A) Top and bottom views of the electrostatic surface for one cross-sectional layer from NLRP6^PYD and ASC^PYD filaments. (B) Computational analysis of the binding energy between the NLRP6^PYD bottom surface and the ASC^PYD top surface and between the ASC^PYD bottom surface and the NLRP6^PYD top surface, showing that the former is much preferred. (C) Per-residue free-energy contribution of the favorable model listed with a cutoff of −1.5 kcal/mol. (D) Detailed interactions of type I, II, and III interfaces in the favorable NLRP6^PYD-ASC^PYD hetero-oligomer model.

Fig. 6.

Full-length NLRP6 assembles into filaments in a concentration-dependent manner. (A) Negative-staining EM image of NLRP6^FL filaments. (B) Cryo-EM image of NLRP6^FL filaments. (C) Power spectrum of aligned NLRP6^FL filament segments from the cryo-EM data. (D) A 2D class of NLRP6^FL filaments. The presumed locations of PYD, NBD, and LRR are shown. (E) Negative-staining EM images of NLRP6^FL at different concentrations. (F) A hypothetical model of NLRP6 inflammasome activation. In brief, full-length NLRP6 may be autoinhibited. On activation, it oligomerizes through both the NBD and the PYD, and the PYD filaments provide the platform for ASC recruitment and oligomerization through PYD–PYD interactions. The CARD in ASC then further oligomerizes and recruits caspase-1, driving caspase-1 dimerization and activation.