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Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism

Nature Structural & Molecular Biology volume 23, pages 416–425 (2016)Cite this article

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Abstract

Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of the human caspase-1 CARD domain (caspase-1CARD) filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins: human inhibitor of CARD (INCA or CARD17) and ICEBERG (CARD18). Our results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on caspase-1CARD polymerization in vitro and inflammasome activation in cells. Whereas caspase-1CARD uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces and thus terminates the caspase-1 filament.

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Figure 1: Cryo-EM structure determination of the caspase-1CARD filament.
Figure 2: Structural analysis of the caspase-1CARD filament.
Figure 3: ICEBERG is filamentous, whereas INCA is monomeric.
Figure 4: INCA inhibits ASCCARD-nucleated formation of caspase-1CARD filaments.
Figure 5: INCA preferentially interacts with caspase-1CARD.
Figure 6: ICEBERG interacts with caspase-1CARD by comixing.
Figure 7: Inducible expression of ICEBERG and INCA on NLRP3-inflammasome activation in modified THP-1 cells treated with LPS and nigericin (nig).
Figure 8: Comparison of critical interface residues between caspase-1CARD and INCA.

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Acknowledgements

The work was supported by US National Institutes of Health (NIH) grants to H.W. (Pioneer Award DP1-HD-087988), to H.L.P. (Pioneer Award DP1-GM-106409), and to Q.Y. (R00: 4R00AI108793-02). F.I.S. was supported by an Advanced Postdoc.Mobility Fellowship from the Swiss National Science Foundation. The cryo-EM facility was funded through the NIH grant AI100645, Center for HIV/AIDS Vaccine Immunology and Immunogen Design (CHAVI-ID). The experiments were performed in part at the Center for Nanoscale Systems at Harvard University, a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the US National Science Foundation (NSF) under award no. ECS-0335765. We thank E. Egelman for generous guidance in methods of helical reconstruction.

Author information

Author notes

  1. Qian Yin

    Present address: Present address: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.,

  2. Alvin Lu and Yang Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Alvin Lu, Yang Li, Qian Yin, Tian-Min Fu, Alexander B Tong & Hao Wu

  2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Alvin Lu, Yang Li, Qian Yin, Tian-Min Fu, Alexander B Tong & Hao Wu

  3. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Florian I Schmidt & Hidde L Ploegh

  4. Center for Quantitative Biology, Peking-Tsinghua Joint Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, Beijing, China

    Shuobing Chen & Youdong Mao

  5. Department of Cancer Immunology and Virology, Intel Parallel Computing Center for Structural Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Shuobing Chen & Youdong Mao

  6. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hidde L Ploegh

  7. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Youdong Mao

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Contributions

A.L., Y.L., F.I.S., Q.Y., S.C., T.-M.F., and Y.M. performed the experiments and analyzed the data. A.L., Q.Y., and T.-M.F. purified the recombinant proteins and performed biochemical experiments. S.C. and Y.M. collected the cryo-EM data, and Y.L. processed the data and completed the helical reconstruction. F.I.S. generated stable cell lines and performed cellular assays, and H.L.P. supervised the experiments. A.B.T. performed Rosetta refinement. A.L. and H.W. conceived the study and wrote the manuscript.

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Correspondence to Hao Wu.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM structure determination of the caspase-1CARD filament.

(a) Enlarged experimental and simulated power spectra showing the position of the meridional layer line. (b-c) A magnified view of the cryo-EM density for helix 1 and helix 4. (d-e) Gold standard and model versus map Fourier shell correlation plots showing the estimated resolution at 4.8 Å. (f) Summary of MolProbity validation report for the caspase-1CARD filament model. (g) Superposition of the caspase-1CARD structure in the filament with the NMR structure of ICEBERG and a homology model of INCA based on the ICEBERG structure.

Supplementary Figure 2 Oligomeric His-GFP-caspase-1CARD-nucleated formation of caspase-1CARD filaments.

Supplementary Figure 3 Comparison with other helical-DD-fold structures.

(a-c) Overall best alignment of the type I, II, and III dimers in caspase-1CARD filament (cyan, magenta, and green) with those in MAVSCARD filament (gray). (d) Structural alignment of caspase-1CARD and MAVSCARD as performed by the Dali server (Holm, L. & Sander, C.,Trends Biochem. Sci. 20, 478-480, 1995). Buried interfacial residues identified by PISA (Krissinel, E. & Henrick, K., J Mol Biol 372, 774-97, 2007) for each asymmetric dimer types were highlighted to show the similarity in their relative locations. (e) Comparison of relative angular differences in the caspase-1CARD filament (cyan, magenta, and green) and in the MAVSCARD filament, the PIDDosome complex, the Myddosome complex, and the ASCPYD filament. The left subunits (Ib, IIb, and IIIb monomers) are first aligned in the respective dimers, and the rotation angles required to bring the Ia, IIa, and IIIa monomers in superposition are indicated.

Supplementary Figure 4 ICEBERG does not interact with ASCCARD.

(a) ICEBERG did not nucleate ASCCARD polymerization. (b) ICEBERG did not inhibit GFP-ASCCARD nucleated ASCCARD polymerization.

Supplementary Figure 5 Gel-filtration profiles of INCA alone, caspase-1CARD-SUMO alone, and a mixture of the two proteins.

INCA does not interact with monomeric caspase-1CARD. Size-exclusion chromatography was done at concentrations comparable to FP assay for caspase-1CARD labeled with TAMRA fluorophore and its mixture with slightly over-stoichiometric amount of INCA.

Supplementary Figure 6 A model of how INCA caps growing caspase-1CARD oligomer.

Conserved type Ia, IIa, IIIa, and IIIb interfaces allow stochastic incorporation of INCA into growing caspase-1CARD filament. Defective type Ib and IIb interfaces prevent caspase-1 recruitment and filament elongation. C1: caspase-1; I: INCA. The composite electrostatic surface and ribbon diagram at the right is complemented with a top view schematic and a side view helical plot at the right. (a) Normal activation. (b) Caspase-1CARD capped by INCA.

Supplementary Figure 7 Effect of R55E mutation on the inhibition potency of INCA.

This charge-reversal mutation at the type Ia interface greatly reduced INCA's inhibition potency on nucleated caspase-1CARD polymerization. Notably, the Ki is increased to ~1.7 μM.

Supplementary Figure 8 Mechanistic model for the inhibition of inflammasome activation by INCA.

Steps: 1. Activation of sensor proteins such as ALRs (e.g. AIM2) and NLRs (e.g. NLRP3) forms an initial PYD layer. 2. The sensor PYDs nucleate the formation of ASCPYD filaments that further recruit ASC monomers. 3. ASCCARD clusters outside of these PYD filament core. 4. Pro-caspase-1 monomers are recruited by CARD-CARD interaction to form CARD filaments. 5a. During normal activation, caspase-1 filaments elongate by recruitment of pro-caspase-1 monomers. 6. Proximity-driven dimerization and autoprocessing lead to the production of active caspase-1 dimers. 5b. In the presence of INCA, capsase-1 filaments are capped to prevent recruitment of pro-caspase-1 thereby repressing inflammasome activation.

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Lu, A., Li, Y., Schmidt, F. et al. Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism. Nat Struct Mol Biol 23, 416–425 (2016). https://doi.org/10.1038/nsmb.3199

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