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Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization

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Science  23 Oct 2015:
Vol. 350, Issue 6259, pp. 404-409
DOI: 10.1126/science.aac5789
  • Fig. 1 Preparation and characterization of NAIP-NLRC4Δ complexes.

    (A) Domain organizations of Salmonella typhimurium PrgJ, mouse NAIP2, mouse NLRC4, and mouse caspase-1. Domain size is drawn approximately to scale; residue numbers are labeled. (B) SDS–polyacrylamide gel electrophoresis (PAGE) of different fractions of the sucrose gradient ultracentrifugation during the purification of the PrgJ-NAIP2-NLRC4Δ complex. Locations of the three component proteins are labeled. The asterisk indicates a contaminating band. (C) A representative negative-stain EM image from fraction 7 in (B). (D) SDS-PAGE of amylose resin elution (lane 1), anti-Flag flow-through (lane 2), and anti-Flag elution (lane 3) fractions during the purification of the coexpressed His-FliC–Flag-NAIP5–His-MBP-NLRC4Δ complex. An enlarged image of lane 3 is shown. (E) Ni-NTA gold labeling (5 nm) of purified His-Sumo-PrgJ–NAIP2ΔBIR-His–His-Sumo-NLRC4Δ complex upon removal of the His-Sumo tag. (F) Schematic diagram of partial and complete inflammasome particles that contain variable ratios between NAIP2 (yellow) and NLRC4 (cyan). (G) Representative cryo-EM micrograph of PrgJ-NAIP2-NLRC4Δ particles. (H) An averaged 2D class of the 11-bladed PrgJ-NAIP2-NLRC4Δ inflammasome complex. The dimensions of the image are 43.5 nm × 43.5 nm.

  • Fig. 2 Cryo-EM structure determination and conformational activation of NLRC4.

    (A) Cryo-EM map of the C11 PrgJ-NAIP2-NLRC4Δ complex colored with local resolution calculated by ResMap using two separately refined half maps. (B) Superimposed ribbon diagram and transparent surface of the C11 NLRC4Δ structure. (C) Cryo-EM density superimposed with one NLRC4Δ subunit. (D) A close-up view of the structure of the NBD of NLRC4Δ superimposed with the cryo-EM density. (E and F) Cryo-EM maps and fitted NLRC4Δ models for the C10 reconstruction at 12.5 Å resolution (E) and the C12 reconstruction at 7.5 Å resolution (F). (G) The WHD-HD2-LRR domain of NLRC4 swings 87.5° to transit from the inactive conformation (left, PDB ID 4KXF) to the active conformation (right). NBD and HD1 are shown in superimposed ribbon diagram and transparent surface, and the WHD-HD2-LRR module is shown in ribbon diagram. (H) Superimposed inactive (colored) and active (gray, except for α14 helix, which is in dark blue) conformations of NLRC4Δ. The α14 helices in the two conformations are labeled to show the relative rotations and the rotational pivot point.

  • Fig. 3 Conformational activation of NLRC4 by activated NAIP2.

    (A) Superposition of the NLRC4Δ structure and the NAIP2ΔBIR homology model in the active conformations. (B) A ribbon diagram of the 11-bladed NAIP2-NLRC4 inflammasome disk, with the single NAIP2 molecule in yellow and NLRC4 molecules in cyan. (C) Locations of A and B surfaces, in particular NAIP2-A and NLRC4-B, in the PrgJ-NAIP2-NLRC4Δ inflammasome. (D) Mapped interactions at the NAIP2-A surface (pink) and the NLRC4-B surface (green) and their surface electrostatic potentials. Dotted ovals show the approximate locations of the interface on the electrostatic surfaces. (E) Detailed interactions between the NAIP2-A surface and the NLRC4-B surface. Those on the A surface are labeled in boldface. (F) Mutations at the NAIP5-A and NLRC4-B surfaces impaired complex formation. The mutated residues in NAIP5 are completely conserved in NAIP2 and NLRC4. The three proteins were coexpressed in 293T cells; the MBP tag was used to pull down the complex, and the component proteins were detected using Western blots. (G) Ribbon diagram of NLRC4Δ in inactive conformation. The region of WHD at the tip of the α14, α15, and α16 helices, in slight clash with an interacting NAIP2, is shown in yellow. Amino acid abbreviations: D, Asp; E, Glu; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; V, Val; Y, Tyr.

  • Fig. 4 NLRC4 polymerization and caspase-1 activation.

    (A) Locations of NLRC4-A and NLRC4-B surfaces. (B) Mapped interactions at NLRC4-A (pink) and NLRC4-B (green) surfaces and the surface electrostatic potentials. (C) Detailed interactions between two neighboring NLRC4 molecules. Those on the A surface are labeled in boldface. (D) Mutations at the NLRC4-A surface impaired NLRC4 recruitment. Three proteins were coexpressed in 293T cells. The Flag tag was used to pull down the complex; component proteins were detected using Western blots. (E) NLRC4CARD, instead of NLRC4FL, nucleates filament formation of labeled caspase-1CARD, as shown by increase in fluorescence polarization. (F) The PrgJ-NAIP2-NLRC4FL inflammasome nucleates filament formation of labeled caspase-1CARD at substoichiometric ratios, as shown by increase in fluorescence polarization. (G) Schematic diagram for mechanism of PrgJ-NAIP2–nucleated polymerization of NLRC4, followed by caspase-1 dimerization and activation.

Supplementary Materials

  • Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization

    Liman Zhang, Shuobing Chen, Jianbin Ruan, Jiayi Wu, Alexander B. Tong, Qian Yin, Yang Li, Liron David, Alvin Lu, Wei Li Wang, Carolyn Marks, Qi Ouyang, Xinzheng Zhang, Youdong Mao, Hao Wu

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S8
    • Table S1

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