Mechanistic insights from inflammasome structures

Abstract

Inflammasomes are supramolecular complexes that form in the cytosol in response to pathogen-associated and damage-associated stimuli, as well as other danger signals that perturb cellular homoeostasis, resulting in host defence responses in the form of cytokine release and programmed cell death (pyroptosis). Inflammasome activity is closely associated with numerous human disorders, including rare genetic syndromes of autoinflammation, cardiovascular diseases, neurodegeneration and cancer. In recent years, a range of inflammasome components and their functions have been discovered, contributing to our knowledge of the overall machinery. Here, we review the latest advances in inflammasome biology from the perspective of structural and mechanistic studies. We focus on the most well-studied components of the canonical inflammasome - NAIP-NLRC4, NLRP3, NLRP1, CARD8 and caspase-1 - as well as caspase-4, caspase-5 and caspase-11 of the noncanonical inflammasome, and the inflammasome effectors GSDMD and NINJ1. These structural studies reveal important insights into how inflammasomes are assembled and regulated, and how they elicit the release of IL-1 family cytokines and induce membrane rupture in pyroptosis.

Fig. 1 |. The canonical and noncanonical inflammasome pathways.

The figure summarizes the canonical inflammasomes comprising NLRP1, NLRP3, NLRP6, NAIP–NLRC4, AIM2 and pyrin, the noncanonical inflammasomes formed by caspase-4 and caspase-5 (human) and caspase-11 (mouse), and the adaptor and effector proteins associated with these pathways, notably the adaptor ASC and the effectors caspase-1, IL-1 family cytokines, GSDMD and NINJ1. Shown are the major protein domains involved in structural organization and function. For more detailed descriptions of each pathway, see the main text. AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a CARD; BIR, baculoviral inhibitor of apoptosis repeat; CARD, caspase recruitment domain; DPP, dipeptidyl peptidase; dsDNA, double-stranded DNA; GSDMD, gasdermin D; HIN, haematopoietic interferon-inducible nuclear; LLPS, liquid–liquid phase separation; LPS, lipopolysaccharide; LRR, leucine-rich repeat; NACHT, nucleotide-binding and oligomerization domain; NAIP, neuronal apoptosis inhibitory protein; NBD, nucleotide-binding domain; NINJ1, ninjurin 1; NLRC, NAIP–NBD-containing, LRR-containing and CARD-containing protein; NLRP, NBD-containing, LRR-containing and PYD-containing protein; PYD, pyrin domain; UVB, ultraviolet B radiation; VbP, Val-boro-pro (DPP8 and DPP9 inhibitor).

Fig. 2 |. The NAIP–NLRC4 inflammasome.

NAIP5, a typical NAIP, and flagellin, a NAIP5 ligand, are used as examples. a, Domain architectures of NAIP5 and NLRC4, with the domain colours matching those shown in part b. Domains that are shown in grey indicate that the structure of this specific region is not shown in part b. b, Formation of the NAIP5–NLRC4 inflammasome. Step 1: the unliganded NAIP5 (Protein Data Bank [PDB:7RAV]) and inactive NLRC4 [PDB:4KXF] at resting state, shown as a weak dimer complex. NAIP5 and NLRC4 can also be monomers before activation. Step 2 and step 3: the interaction between bacterial flagellin and NAIP5 induces the activation of NAIP5 [PDB:5YUD] and NLRC4 [PDB:4KXF], together with conformational changes. The grey NLCR4 molecule in step 3 represents the inactive state before its conformational changes to the active state. Step 4 to step 6: the activated NLRC4 self-propagates [PDB:6B5B], building up an inflammasome disc [PDB:3JBL]. Domains are colour coded as in part a. NAIP2 follows the same mechanism in forming an inflammasome disc. BIR, baculoviral inhibitor of apoptosis repeat; CARD, caspase recruitment domain; HD, helical domain; ID, intermediate domain; LRR, leucine-rich repeats; NAIP, neuronal apoptosis inhibitory protein; NBD, nucleotide-binding domain; NLRC, NAIP–NBD-containing, LRR-containing and CARD-containing protein; NTD, N-terminal domain; WHD, winged helix domain.

Fig. 3 |. The NLRP3 inflammasome.

a, Domain architectures of NLRP3, NEK7 and ASC, with the domain colours matching those shown in part b. Typical disease-associated mutations in NLRP3 are highlighted. b, Formation of the NLRP3 inflammasome. Step 1: upon upregulation of their expression (‘priming’), NLRP3 proteins are present in the cytosol and on the trans-Golgi network (TGN) as monomeric proteins or inactive ‘cage’ structures [PDB:7PZC]. Step 2: upon stimulation, inactive NLRP3 cages presumably undergo conformational changes. The TGN disperses into vesicles (dispersed TGN (dTGN)) containing hypothetical active NLRP3 cages. Step 3: via microtubule trafficking, the vesicles move to the microtubule organizing centre (MTOC), wherein NEK7 [PDB:6S76] proteins are located. Step 4 and step 5: NEK7 interacts with NLRP3 cages [PDB:6NPY], presumably disrupting and opening each cage into two halves. Step 6: the two halves rearrange and unite into an inflammasome disc [PDB:8EJ4 and PDB:8ERT]. Step 7 and step 8: ASC [PDB:2KN6] adaptor proteins are recruited to the NLRP3 inflammasome disc via homotopic PYD–PYD interactions [PDB:8EJ4, PDB:8ERT and PDB:3J63], resulting in a PYD–PYD filament that oligomerizes the CARDs of ASC molecules to mediate caspase-1 recruitment. Domains are colour coded as in part a. ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase recruitment domain; FISNA, fish-specific NACHT-associated domain; HD, helical domain; LRR, leucine-rich repeats; NACHT, nucleotide-binding and oligomerization domain; NBD, nucleotide-binding domain; NLRP, NBD-containing, LRR-containing and PYD-containing protein; PYD, pyrin domain; WHD, winged helix domain.

Fig. 4 |. The NLRP1 inflammasome.

a, Domain architectures of NLRP1 and DPP9. Domains that are shown in grey indicate that the structure of this specific region is not shown in parts b–e. The unlabelled domains in NLRP1 represent predicted unstructured regions. Typical disease-associated mutations in NLRP1 are highlighted. The proteolysis site on the FIIND is marked as a gap. b, The structure of an NLRP1 and DPP9 ternary complex, a dimeric complex in which each half of the dimer contains ZU5 and UPA (the FIIND) of full-length NLRP1, the UPA of a processed NLRP1 C-terminal fragment and a DPP9 monomer [PDB:6X6A]. The N-terminal end of the NLRP1 C-terminal UPA inserts into the active-site tunnel of DPP9. c, The structure of DPP9 bound with the small-molecule inhibitor Val-boro-pro (VbP) in complex with NLRP1 [PDB:6X6C]. VbP occupies the active site of DPP9, which releases the NLRP1 C-terminal UPA, allowing for the activation of NLRP1. d, Clustering of NLRP1 UPA [PDB:6XKK] promotes the homotypic interactions of NLRP1 CARDs to form filaments. e. Two different orientations of an NLRP1 CARD filament [PDB:6K7V]. For parts b–e, domains are colour coded as in part a. CARD, caspase recruitment domain; CT, C-terminus; DPP, dipeptidyl peptidase; FIIND, function-to-find domain; HD, helical domain; LRR, leucine-rich repeats; NBD, nucleotide-binding domain; NLRP, NBD-containing, LRR-containing and PYD-containing protein; NT, N-terminus; PYD, pyrin domain; WHD, winged helix domain.

Fig. 5 |. Gasdermins.

a, Domain architectures of a gasdermin and caspase-1. Domains that are shown in grey indicate that the structure of this specific region is not shown in parts b–g. b, A gasdermin molecule, represented using the structure of mouse GSDMA3 [PDB:5B5R]. The scissor icon shows the interdomain cleavage site of gasdermin. c, The structure of caspase-1 p20/p10 dimer in complex with GSDMD [PDB:6VIE and 6KMV]. The active sites are marked as scissor icons, and the exosites are marked by circles. d, The structural changes of a human GSDMD N-terminal subunit from autoinhibited state [PDB:6N9O] to pore-forming state [PDB:6VFE], which could occur during prepore to pore transition on the membrane. The pore-forming state of GSDMD N-terminal fragment looks like a stretched hand; as the ‘fingers’ stretch out to form the transmembrane β-barrel, the globular domain undergoes a 38° rotation. e, The structure of a 33-subunit GSDMD N-terminal pore viewed from two orientations [PDB:6VFE]. f, Superposition of human GSDMD (blue) and human GSDMB (purple; [PDB:8ET2 and 8GTN]) pore-forming subunits and the structure of a 24-subunit GSDMB N-terminal pore viewed from two orientations [PDB:8ET2]. g, Electrostatic surface of the GSDMD pore viewed from outside and inside [PDB:6VFE]. The GSDMD membrane-binding regions are positively charged (blue), whereas the GSDMD conduit is negatively charged (red). The cargo release route is marked by an arrow. For parts b–f, domains are colour coded as in part a. CDL, CARD linker; CT, C-terminus; IDL, interdomain linker; NT, N-terminus.

Fig. 6 |. NINJ1.

a, Domain architecture of ninjurin 1 (NINJ1). The domain shown in grey indicates that the structure of this specific region is not shown in parts b–f. b, Structural presentations of a NINJ1 double filament [PDB:8CQR] (left) and two stacked NINJ1 subunits (right). The double filament contains six subunits each with a different shade of blue or pink. Secondary structures are labelled for the blue subunit to the right. c, Structure of a single NINJ1 subunit. d, A single filament of five NINJ1 subunits (modelled from [PDB:8CQR]), in two orientations. The α1 helix of one subunit extends over to the transmembrane helices α3 and α4 of a neighbouring subunit to make a NINJ1 chain. e, Structural representation of a NINJ1 subunit from rings or curved filaments, showing bent helices α3 and α4. f, A model of a partial NINJ1 ring built by propagating the NINJ1 segment structure, showing the concave hydrophobic side and the convex hydrophilic side. g, Two alternative models of NINJ1 activation: the membrane damage model, in which NINJ1 filaments or pores cause membrane leakage, and the membrane loss model, in which NINJ1 assembles around a patch of membrane, leading to its release. In both models, the α1 and α2 subunits of NINJ1 (shown in yellow) directly cause membrane damage. TM, transmembrane helix.