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Cryo-EM structures of the active NLRP3 inflammasome disc

Abstract

Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers1,2,3,4,5, and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing protein  3 (NLRP3) is an inflammasome sensor of membrane damage highly important in regard to the induction of inflammation2,6,7. Here we report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with adenosine 5′-O-(3-thio)triphosphate, the centrosomal NIMA-related kinase 7 (NEK7) and the adaptor protein ASC, which recruits caspase-1. In these NLRP3–NEK7–ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by about 85° relative to the ADP-bound inactive conformation8,9,10,11,12. The fish-specific NACHT-associated domain conserved in NLRP3 but absent in most NLRPs13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disc. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all NLRP3 subunits combine to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disc interfaces. Together with previous structures of an inactive NLRP3 cage in which LRR–LRR interactions play an important role8,9,10,11, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disc.

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Fig. 1: Preparation and characterization of the NLRP3–NEK7–ASC inflammasome complex.
Fig. 2: Cryo-EM structure of the C10 NLRP3–NEK7–ASC inflammasome complex.
Fig. 3: Conformational activation of the NLRP3 inflammasome.
Fig. 4: Interaction surfaces in the NLRP3 oligomer.
Fig. 5: A structure-derived model of NLRP3 inflammasome activation.

Data availability

Atomic coordinates of the tenfold NLRP3–NEK7 assembly structure and NLRP3 PYD filament have been deposited in PDB under accession nos. 8EJ4 and 8ERT, respectively. Corresponding cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession nos. EMD-28175 and EMD-28560, respectively. All other data are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank H. Sharif, L. Andreeva, M. Mahammad, L. Wang and L. David for earlier work on this project, and Wu laboratory members for useful discussions. We thank Z. Yang for making lentiviruses and reconstitution of NLRP3 mutants into NLRP3–/– iBMDMs. For cryo-EM data collection we thank R. Walsh, S. Sterling, M. Megan, S. Rawson and Z. Li at the Harvard Cryo-EM Center for Structural Biology, and K. Song and J. Chang at the Cryo-EM Core Facility at University of Massachusetts Medical School. We thank K. Fitzgerald at University of Massachusetts Medical School for NLRP3–/– iBMDMs. We also thank R. Tomaino and Taplin Biological Mass Spectrometry Facility for protein analysis. Our research used software and computing support at SBGrid. This work was supported by US National Institutes of Health (nos. R01AI124491 to H.W. and R21AR079766 to V.G.M.), a postdoctoral fellowship from the KidneyCure Foundation (to L.X.) and a Faculty Career Development Fellowship from OFD/BTREC/CTREC Program of Boston Children’s Hospital (to V.G.M.).

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Authors and Affiliations

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Contributions

H.W. and L.X. conceived the study. V.G.M. generated and carried out initial expression of NLRP3 and NEK7 constructs by immunoblotting. L.X. designed and performed all experiments and H.W. supervised the project. L.X. and H.W. wrote the manuscript.

Corresponding author

Correspondence to Hao Wu.

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H.W. is a cofounder of Ventus Therapeutics. L.X. and V.G.M. declare no competing interests.

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Nature thanks Edward Miao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Preparation and characterization of the NLRP3-NEK7 complex.

a, Gel filtration profile of the NLRP3-NEK7 complex showing the two peaks. b, SDS-polyacrylamide gel electrophoresis (PAGE) of gel filtration fractions for the NLRP3-NEK7 complex. Locations of the two component proteins are labeled. c, Representative negative-staining images from the two NLRP3-NEK7 complex peaks. d, Gel filtration profile of the NLRP3-NEK7 complex with ATPγS showing one major peak. e, SDS-PAGE of the gel filtration fractions for the NLRP3-NEK7 complex with ATPγS. Locations of the two component proteins are labeled. f, A representative negative-staining image from the peak containing NLRP3, NEK7 and ATPγS. g, A representative cryo-EM image from the peak containing NLRP3, NEK7 and ATPγS. Experiments in (a-g) were repeated at least three times. h, 2D classes from cryo-EM images of the NLRP3-NEK7 complex with ATPγS. No full disk was observed.

Extended Data Fig. 2 Preparation and characterization of NLRP3-NEK7-ASC inflammasome complexes.

a, Gel filtration profile of the NLRP3-NEK7-ASC complex. b, SDS- PAGE of the near void peak from the gel filtration chromatography. Locations of the three component proteins are labelled. c, Mass spectrometry that detected ASC PYD in the band in (b). d, A representative cryo-EM image from a sample containing NLRP3, NEK7, ATPγS, and 1:1 ASC PYD. Experiments in (a-b) and (d) were repeated at least three times.

Extended Data Fig. 3 Flow chart for cryo-EM data processing of the NLRP3-NEK7-ASC inflammasome complex.

Data processing details can be found in Methods.

Extended Data Fig. 4 Local resolution distributions and Fourier shell correlation (FSC) curves.

a-c, The C10 NLRP3-NEK7-ASC complex (a), the C11 NLRP3-NEK7-ASC complex (b), and the partial NLRP3-NEK7 disk containing primarily 5 subunits (c).

Extended Data Fig. 5 PYD helical filament.

a-c, Side views of the cryo-EM maps of the C10 NLRP3-NEK7-ASC complex (a), the C11 NLRP3-NEK7-ASC complex (b) and the partial disk of the NLRP3-NEK7 complex (c). d, Local resolution distribution and Fourier shell correlation (FSC) curve of the NLRP3 PYD filament.

Extended Data Fig. 6 Close-up evaluation of the C10 NLRP3-NEK7-ASC complex map quality.

a-h, Cryo-EM maps in individual regions (labelled) superimposed with the final model.

Extended Data Fig. 7 Conformational change and comparison with NLRC4.

a, Superposition of active NLRP3 (in domain colours) and inactive NLRP3 (PDB: 6NPY in grey) by the FISNA-NBD-HD1 domain. The WHD domain of inactive NLRP3 situates behind the superimposed HD1, different from that of active NLRP3. b, Superposition of active NLRP3 and inactive NLRP3 (PDB: 6NPY in grey), showing the LRR domain and NEK7 of the inactive NLRP3-NEK7 complex would have been in clash with a neighbouring NLRP3 molecule. c, Superposition of active NLRP3 with active NLRC4 (PDB: 3JBL in yellow) by the FISNA-NBD-HD1 domain. d, Superposition of the FISNA domain from active NLRP3 (salmon), inactive NLRP3 (PDB: 6NPY in green), active NLRC4 (PDB: 3JBL in cyan), inactive NLRC4 (PDB: 4KXF in grey), and active NAIP5 (6B5B in yellow).

Extended Data Fig. 8 Mapping of the CAPS mutations on active NLRP3, and comparison of active NLRP3 and NLRC4.

a, Locations of CAPS mutation sites on the active NLRP3 structure. Two views are shown. b, A list of the mutations, their domain location and potential structural effects. c, Superposition of the WHD from active NLRP3 (magenta) and inactive NLRP3 (PDB: 6NPY in grey), showing the formation of a β-hairpin in the active state. d, Superposition of the WHD from active NLRP3 (magenta) and active NLRC4 (PDB: 3JBL in yellow), showing lack of the β-hairpin in NLRC4. e, Superposition of one NLRP3 subunit in two neighbouring NLRP3 subunits in a disk (coloured by domains) with one NLRC4 subunit in two neighbouring NLRC4 subunits in a disk (PDB: 3JBL in yellow). The second NLRC4 subunit needs to rotate by 31.7° to align with the second NLRP3 subunit.

Extended Data Fig. 9 A structure-derived model for potential NEK7-independent, and LRR-deleted NLRP3 inflammasome activation.

a, Under certain conditions such as those marked in the schematic (e.g. high NLRP3 expression level or certain effects from priming), monomeric NLRP3 or destabilized caged NLRP3 may directly form the active NLRP3 disk with ASC upon stimulation. b, For overexpressed LRR-deleted NLRP3, stimulation would likely directly induce the activating conformational change to allow the assembly of the inflammasome disk with ASC.

Extended Data Table 1 Cryo-EM structure determination

Supplementary information

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Xiao, L., Magupalli, V.G. & Wu, H. Cryo-EM structures of the active NLRP3 inflammasome disc. Nature 613, 595–600 (2023). https://doi.org/10.1038/s41586-022-05570-8

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