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Mechanism of NAIP—NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5

Nature Structural & Molecular Biology volume 30, pages 159–166 (2023)Cite this article

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An Author Correction to this article was published on 25 January 2023

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Abstract

The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP—NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17–18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5–NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation.

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Fig. 1: Biochemical purification and cryo-EM structure of NAIP5.
Fig. 2: ATP binding and the conformational change on NAIP5 activation.
Fig. 3: Structural basis of ligand-induced NAIP5 activation.
Fig. 4: NAIP5 activation and NAIP5-triggered NLRC4 activation.

Data availability

The cryo-EM maps were deposited in the Electron Microscopy Data Bank under the accession IDs EMD-24387 (3.3 Å) and EMD-24389 (3.6 Å), and the atomic coordinates were deposited in the PDB under the accession ID 7RAV. Plasmids are available from the corresponding author. Source data are provided with this paper.

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References

  1. Harton, J. A., Linhoff, M. W., Zhang, J. & Ting, J. P. Cutting edge: CATERPILLER: a large family of mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains. J. Immunol. 169, 4088–4093 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Proell, M., Riedl, S. J., Fritz, J. H., Rojas, A. M. & Schwarzenbacher, R. The Nod-like receptor (NLR) family: a tale of similarities and differences. PLoS ONE 3, e2119 (2008).

  3. Rauch, I. et al. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo. J. Exp. Med. 213, 657–665 (2016).

  4. Kofoed, E. M. & Vance, R. E. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477, 592–595 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhao, Y. et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477, 596–600 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Yang, J., Zhao, Y., Shi, J. & Shao, F. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation. Proc. Natl Acad. Sci. USA 110, 14408–14413 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Reyes Ruiz, V. M. et al. Broad detection of bacterial type III secretion system and flagellin proteins by the human NAIP/NLRC4 inflammasome. Proc. Natl Acad. Sci. USA 114, 13242–13247 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Duncan, J. A. & Canna, S. W. The NLRC4 inflammasome. Immunological Rev. 281, 115–123 (2018).

    Article  CAS  Google Scholar 

  9. Bauer, R. & Rauch, I. The NAIP/NLRC4 inflammasome in infection and pathology. Mol. Asp. Med. 76, 100863 (2020).

    Article  CAS  Google Scholar 

  10. Miao, E. A. et al. Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc. Natl Acad. Sci. USA 107, 3076–3080 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. Miao, E. A. et al. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat. Immunol. 7, 569–575 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Hu, Z. et al. Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science 341, 172–175 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Hu, Z. et al. Structural and biochemical basis for induced self-propagation of NLRC4. Science 350, 399–404 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, L. et al. Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science https://doi.org/10.1126/science.aac5789 (2015).

  16. Wu, H. Higher-order assemblies in a new paradigm of signal transduction. Cell 153, 287–292 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, Y. et al. Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1. Proc. Natl Acad. Sci. USA 115, 10845–10852 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tenthorey, J. L. et al. The structural basis of flagellin detection by NAIP5: a strategy to limit pathogen immune evasion. Science 358, 888–893 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yang, X. et al. Structural basis for specific flagellin recognition by the NLR protein NAIP5. Cell Res. 28, 35–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Zhang, L. & Wu, H. Bad germs are trapped. Cell Res. 28, 141–142 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Canna, S. et al. A157: macrophage activation syndrome-like illness due to an activating mutation in NLRC4. Arthritis Rheumatol. 66, S203 (2014).

    Article  Google Scholar 

  22. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D., Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  26. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D. Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Halff, E. F. et al. Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin. J. Biol. Chem. 287, 38460–38472 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tenthorey, J. L., Kofoed, E. M., Daugherty, M. D., Malik, H. S. & Vance, R. E. Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes. Mol. Cell. https://doi.org/10.1016/j.molcel.2014.02.018 (2014).

  29. Poyet, J. L. et al. Identification of Ipaf, a human caspase-1-activating protein related to Apaf-1. J. Biol. Chem. 276, 28309–28313 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. White, S. R. & Lauring, B. AAA+ ATPases: achieving diversity of function with conserved machinery. Traffic 8, 1657–1667 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Maharana, J., Panda, D. & De, S. Deciphering the ATP-binding mechanism(s) in NLRP-NACHT 3D models using structural bioinformatics approaches. PLoS ONE 13, e0209420 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Maekawa, S., Ohto, U., Shibata, T., Miyake, K. & Shimizu, T. Crystal structure of NOD2 and its implications in human disease. Nat. Commun. 7, 11813 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yuan, S. & Akey, C. W. Apoptosome structure, assembly, and procaspase activation. Structure 21, 501–515 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pang, Y. et al. Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila. Genes Dev. 29, 277–287 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Sharif, H. et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature https://doi.org/10.1038/s41586-019-1295-z (2019).

  36. Wang, J. et al. Reconstitution and structure of a plant NLR resistosome conferring immunity. Science https://doi.org/10.1126/science.aav5870 (2019).

  37. Wang, J. et al. Ligand-triggered allosteric ADP release primes a plant NLR complex. Science 364, eaav5868 (2019).

  38. Erijman, A., Dantes, A., Bernheim, R., Shifman, J. M. & Peleg, Y. Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J. Struct. Biol. 175, 171–177 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  40. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Preprint at bioRxiv https://doi.org/10.1101/2020.06.12.148296 (2020).

  41. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D. Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bryan, N. B., Dorfleutner, A., Rojanasakul, Y. & Stehlik, C. Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J. Immunol. 182, 3173–3182 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Li, J., Yin, H. L. & Yuan, J. Flightless-I regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity. J. Cell Biol. 181, 321–333 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Lopez at the Multiscale Microscopy Core of OHSU, S. Mulligan, N. Meyer and C. Yoshioka at the Pacific Northwest Center for Cryo-EM (PNCC) for their help in cryo-EM data collection. A portion of this research was supported by National Institutes of Health grant no. U24GM129547 and performed at the PNCC at OHSU and accessed through the Environmental Molecular Sciences Laboratory (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We thank I. Rauch at OHSU for discussion and X. Xiao at OHSU for sharing the HEK293T cell line. This work was supported by the National Institutes of Health grant nos. R00AI137300 and R01AI165580 (L.Z.), and the Medical Research Foundation New Investigator grant no. 1019214 (L.Z.). We apologize to authors whose work could not be cited because of space limitation.

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Author notes

  1. These authors contributed equally: Bhaskar Paidimuddala, Jianhao Cao.

Authors and Affiliations

  1. Department of Chemical Physiology and Biochemistry, Oregon Health and Science University (OHSU), Portland, OR, USA

    Bhaskar Paidimuddala, Jianhao Cao, Grady Nash, Qing Xie & Liman Zhang

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Hao Wu

  3. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Hao Wu

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  1. Bhaskar Paidimuddala
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Contributions

L.Z., B.P. and J.C. conceived the study. B.P. purified the protein and performed negative stain EM analysis. J.C. made cryo-grids and collected cryo-EM data. L.Z. and J.C. performed data processing. L.Z. performed initial model building, B.P. and Q.X. performed additional refinement. L.Z. and B.P. designed mutants for functional assays, B.P. performed biochemical assays, G.N. did technical replicates. L.Z., B.P., J.C., G.N. and H.W. wrote the manuscript.

Corresponding author

Correspondence to Liman Zhang.

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Competing interests

H.W. is a cofounder of Ventus Therapeutics. The other authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Jun Ma, Edward Miao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Superdex-200 profile of the recombinant Flag-NAIP5 and Negative stain EM for void and monomeric fractions.

a) Size exclusion chromatography profile of Flag-NAIP5; b) SDS-PAGE for fractions from A). Densitometric analysis was performed using Bio-Rad Image Lab Software 6.1 to calculate the fraction of 11.5 ml peak; This experiment was repeated three independent times. c) Negative stain EM image for the Superdex200 fraction eluted from the void peak as shown in B); d) Negative stain EM image for the 11.5 mL Superdex200 fraction as shown in B); e) 2D class averages of particles picked from D). f) Repetition of Fig. 2e with separately purified batches of protein showing FliC does not enhance the ATPase activity of NAIP5.

Source data

Extended Data Fig. 2 Cryo-EM data processing and reconstruction.

a) Schematic workflow of 2D/3D classification and reconstruction. In total, we completed 6 rounds of 3D classification interleaved with 2D classification in each 3D class. After each round of 3D and 2D classification, bad classes were rejected by visual inspection. We obtained 159,513 particles for final refinement, with 36,523 particles from the tilted dataset and 122,990 particles from the un-tilted dataset; b) The gold-standard Fourier Shell Correlation (FSC) plots of the cryo-EM map obtained from non-uniform refinement. FSC curve for the cross-validation of the atomic models of the inactive NAIP5 is also showed; c) The EM density maps of the Non-uniform refinement and Homogeneous refinement with color coded to show the local resolution as calculated by cryoSPARC. d) Orientation distribution map of Non-uniform and Homogeneous refinement. e) Plots of the global half-map FSC (solid red line) together with the spread of directional resolution values (green area encompassed by dotted green lines, left axis), and histogram of directional FSC (blue bars).

Extended Data Fig. 3 Representative EM densities of pre-liganded NAIP5.

EM densities and their corresponding models are shown for the individual domains of pre-liganded NAIP5.

Extended Data Fig. 4 NAIP5 depended NLRC4 activation.

a) the nucleating surface (blue) and receptor surface (red) in the active NLRC4; b) The nucleating surface (blue) and receptor surface (red) in the inactive NLRC4; c) The exposed nucleating surface (blue) on ligand-bound NAIP5; d) The exposed nucleating surface (blue) on pre-liganded NAIP5; e) NLRC4 is recruited to NAIP5 by NAIP5- nucleating surface, and NLRC4- nucleating surface is exposed upon NLRC4 activation to initiate a domino-like process to form inflammasome complex; f) Only in the presence of ligand (FliC-D0L, the smallest FliC fragment effective in activating NAIP5), NAIP5 is able to induce NLRC4 oligomerization, or form a complex with 1 subunit of NLRC4 nucleation surface mutant (NSM), which is deficient in further oligomerization. This experiment was repeated three independent times.

Source data

Extended Data Fig. 5 Sequence alignment of the key regions discussed in this work.

Conserved and similar residues are highlighted in white and red. The blue circles indicate residues tested by mutagenesis. Amino acid numbering corresponds to NAIP5 was given on the top of the alignment.

Supplementary information

Supplementary Information

Primers used in this study.

Reporting Summary

Peer Review File

Supplementary Video 1

The process of NAIP5 and NLRC4 activation.

Source data

Source Data Fig. 1

MALS raw data.

Source Data Fig. 1

Unprocessed western blots and/or gels.

Source Data Fig. 2

ATPase assay raw data.

Source Data Fig. 2

Unprocessed western blots and/or gels.

Source Data Fig. 3

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 1

ATPase assay raw data.

Source Data Extended Data Fig. 1

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 4

Unprocessed western blots and/or gels.

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Paidimuddala, B., Cao, J., Nash, G. et al. Mechanism of NAIP—NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5. Nat Struct Mol Biol 30, 159–166 (2023). https://doi.org/10.1038/s41594-022-00889-2

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