Structural and mechanistic elucidation of inflammasome signaling by cryo-EM

Abstract

The innate immune system forms an evolutionarily ancient line of defense against invading pathogens and endogenous danger signals. Within certain cells of innate immunity, including epithelial cells and macrophages, intricate molecular machineries named inflammasomes sense a wide array of stimuli to mount inflammatory responses. Dysregulation in inflammasome signaling leads to a wide range of immune disorders such as gout, Crohn's disease, and sepsis. Recent technological advances in cryo-electron microscopy (cryo-EM) have enabled the structural determination of several key signaling molecules in inflammasome pathways, from which macromolecular assembly emerges as a common mechanistic theme. Through the assembly of helical filaments, symmetric disks, and transmembrane pores, inflammasome pathways employ highly dynamic yet ordered processes to relay and amplify signals. These unprecedentedly detailed views of inflammasome signaling not only revolutionize our understanding of inflammation, but also pave the way for the development of therapeutics against inflammatory diseases.

Figure 1.. Molecular components and filamentous structures in inflammasome signaling.

(a) Domain organization of proteins involved in inflammasome pathways. (b) Raw cryo-EM images and structures of ASC^PYD (PDB ID: 3J63) and caspase-1^CARD (PDB ID: 5FNA) filaments overlaid with their cryo-EM maps. (c) General procedures of helical processing. For data processing in RELION, in the manual picking step, black circles indicate the start-end coordinates, and black lines indicate the length of the picked filaments. In the auto-picking step, the distance between different black circles is defined as the inter-box distance, which equals the number of asymmetric units multiplied by the helical rise. Good 2D class averages are selected to generate layer line information (He and Scheres 2017). For the IHRSR method in SPIDER, helical filaments are picked in box manually with EMAN (Ludtke, Baldwin, and Chiu 1999). In the segmentation step, the rectangle box length is determined empirically (Egelman 2010). The image source of the powerspectrum (Shen et al. 2019).

Figure 2.. Nucleated polymerization of the NAIP-NLRC4 inflammasome.

(a) A cryo-EM micrograph shows disk-like PrgJ-NAIP2-NLRC4_ΔCARD inflammasome particles boxed and 11- and 12-mer 2D class averages (Zhang et al. 2015). (b) Auto-inhibited (PDB ID: 4KXF) and active conformations of NLRC4_ΔCARD. The latter is a single subunit from the cryo-EM structure of the disk-like PrgJ-NAIP2-NLRC4_ΔCARD inflammasome complex (PDB ID: 6B4B, 3JBL). (c) Mechanism of FlaA recognition within a partial FlaA-NAIP5-NLRC4_ΔCARD inflammasome disk (PDB ID: 6B5B). (d) The whole process of NLRC4_ΔCARD inflammasome activation from a single FlaA-bound NAIP5 to a final 11-subunit disk (PDB ID: 6B4B, 3JBL, 6B5B). (e) Cryo-ET map of the FliC-D0_L-NAIP5-NLRC4 inflammasome (EMDB ID: 2901), showing a spiral architecture with a central CARD filament.

Figure 3.. Molecular mechanism of GSDM pore formation.

(a) Crystal structure of mouse GSDMA3 (PDB ID: 5B5R) with key secondary structure elements labeled, GSDMA3-NT colored in blue, cyan, and yellow, and GSDMA3-CT colored in magenta. (b) A cryo-EM micrograph of detergent-solubilized GSDMA3 pores and 2D class averages of the pores generated in RELION (Ruan et al. 2018; Scheres 2012). (c) Cryo-EM structure of the 27-fold symmetric GSDMA3 membrane pore (PDB ID: 6CB8) with dimensions indicated and a magnified view of a pore-form GSDMA3-NT subunit. Color schemes follow those of the crystal structure in (a). (d) A model for pore formation by the GSDM family, where GSDM-NTs might oligomerize into a membrane-associated pre-pore before insertion of the β-barrel to form a transmembrane pore that allows the passage of cytoplasmic contents such as IL-1β.

Figure 4.. Structural biology of inflammasome signaling.

Overview of the inflammasome activation processes for NLRP3, NLRC4, and AIM2, with domains and proteins indicated above, and the stimuli and cartoon models of NLRP3, NLRC4, and AIM2 inflammasomes shown in boxes. NLRP3 and AIM2 require the adaptor protein ASC for proximity-induced activation of caspase-1. By contrast, NLRC4 can directly recruit and activate caspase-1. Active caspase-1 cleaves and releases the auto-inhibition of GSDMD. GSDMD-NT then forms membrane pores to induce pyroptosis. Active caspase-1 also cleaves pro-IL-1β into its mature form, which is released through GSDMD pores.