Article | Published:

Pore-forming activity and structural autoinhibition of the gasdermin family

Nature volume 535, pages 111116 (07 July 2016) | Download Citation

  • An Erratum to this article was published on 05 October 2016

This article has been updated

Abstract

Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.

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Change history

  • 10 June 2016

    An error in the Reviewer Information section was corrected.

Accessions

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors of GSDMA3 have been deposited in the Protein Data Bank under the accession code 5B5R.

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Acknowledgements

We thank W. Wei for reagents, H. Wang for suggestions on electron microscopy data analysis, and the staff of beamlines BL18U1 and BL19U1 at National Center for Protein Sciences, Shanghai, and Shanghai Synchrotron Radiation Facility for X-ray data collection. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202), the China National Science Foundation Program for Distinguished Young Scholars (31225002) and Program for International Collaborations (31461143006), and the National Basic Research Program of China 973 Program (2012CB518700 and 2014CB849602) to F.S. The research was also supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute and the Beijing Scholar Program to F.S.

Author information

Affiliations

  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    • Jingjin Ding
    • , Yang She
    • , Da-Cheng Wang
    •  & Feng Shao
  2. National Institute of Biological Sciences, Beijing, 102206, China

    • Jingjin Ding
    • , Kun Wang
    • , Wang Liu
    • , Yang She
    • , Qi Sun
    • , Jianjin Shi
    • , Hanzi Sun
    •  & Feng Shao
  3. Foshan University, Guangdong, 528000, China

    • Da-Cheng Wang
  4. National Institute of Biological Sciences, Collaborative Innovation Center for Cancer Medicine, Beijing, 102206, China

    • Feng Shao

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Contributions

J.D., D.-C.W. and F.S. conceived the study; J.D., together with K.W., designed and performed the majority of the experiments; Y.S. helped with protein purification; W.L. performed the pyroptosis assay; Q.S. assisted J.D. in electron microscopy studies; J.S. provided critical reagents and suggestions; H.S. performed structural modelling; and J.D. and F.S. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Da-Cheng Wang or Feng Shao.

Reviewer Information Nature thanks F. Sigworth and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains the uncropped immunoblot images presented in figure 5 and the Extended Data Figures.

Videos

  1. 1.

    Membrane targeting of the Gasdermin-N domain of GSDMD during pyroptosis

    The Gasdermin-N domain of GSDMD (GSDMD-N L192D) fused N-terminal to EGFP was stably expressed in HeLa cells under a tetracycline-inducible promoter. Pyroptosis was triggered by doxycycline addition to induce GSDMD-N L192D-EGFP expression. Shown are real-time videos of two representative pyroptotic cells. Scale bar, 15 μm. The time-lapse images for Video 1 are in Fig. 1c.

  2. 2.

    Membrane targeting of the Gasdermin-N domain of GSDMD during pyroptosis

    The Gasdermin-N domain of GSDMD (GSDMD-N L192D) fused N-terminal to EGFP was stably expressed in HeLa cells under a tetracycline-inducible promoter. Pyroptosis was triggered by doxycycline addition to induce GSDMD-N L192D-EGFP expression. Shown are real-time videos of two representative pyroptotic cells. Scale bar, 15 μm.

  3. 3.

    Membrane targeting of the Gasdermin-N domain of GSDMA during pyroptosis.

    The Gasdermin-N domain of GSDMA3 (GSDMA3-N L184D) fused N-terminal to EGFP was stably expressed in HeLa cells under a tetracycline-inducible promoter. Pyroptosis was triggered by doxycycline addition to induce GSDMA3-N L184D-EGFP expression. Shown are real-time videos of two representative pyroptotic cells. Scale bar, 15 μm. The time-lapse images for Video 3 are in Extended Data Fig. 3c.

  4. 4.

    Membrane targeting of the Gasdermin-N domain of GSDMA during pyroptosis.

    The Gasdermin-N domain of GSDMA3 (GSDMA3-N L184D) fused N-terminal to EGFP was stably expressed in HeLa cells under a tetracycline-inducible promoter. Pyroptosis was triggered by doxycycline addition to induce GSDMA3-N L184D-EGFP expression. Shown are real-time videos of two representative pyroptotic cells. Scale bar, 15 μm.

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https://doi.org/10.1038/nature18590

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