Abstract
Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein1,2, to trigger target cell pyroptosis3. GSDMB and the charter gasdermin family member GSDMD4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently6,8. Here we report the crystal structure of the IpaH7.8–GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins9,10. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies6,8. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.
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References
Broz, P., Pelegrin, P. & Shao, F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol. 20, 143–157 (2020).
Shi, J., Gao, W. & Shao, F. Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem. Sci. 42, 245–254 (2017).
Zhou, Z. et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 368, eaaz7548 (2020).
Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).
Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).
Hansen, J. M. et al. Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions. Cell 184, 3178–3191 (2021).
Luchetti, G. et al. Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection. Cell Host Microbe 29, 1521–1530 (2021).
Rana, N. et al. GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis. Cell 185, 283–298 (2022).
Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016).
Liu, Z. et al. Crystal structures of the full-length murine and human gasdermin D reveal mechanisms of autoinhibition, lipid binding, and oligomerization. Immunity 51, 43–49 (2019).
Jorgensen, I. & Miao, E. A. Pyroptotic cell death defends against intracellular pathogens. Immunol. Rev. 265, 130–142 (2015).
Man, S. M., Karki, R. & Kanneganti, T. D. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 277, 61–75 (2017).
Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).
Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014).
Rathinam, V. A. K., Zhao, Y. & Shao, F. Innate immunity to intracellular LPS. Nat. Immunol. 20, 527–533 (2019).
Liu, X. et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535, 153–158 (2016).
Das, S. et al. GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation. Proc. Natl Acad. Sci. USA 113, 13132–13137 (2016).
Panganiban, R. A. et al. A functional splice variant associated with decreased asthma risk abolishes the ability of gasdermin B to induce epithelial cell pyroptosis. J. Allergy Clin. Immunol. 142, 1469–1478 (2018).
Wang, K. et al. Structural mechanism for GSDMD targeting by autoprocessed caspases in pyroptosis. Cell 180, 941–955 (2020).
Rohde, J. R., Breitkreutz, A., Chenal, A., Sansonetti, P. J. & Parsot, C. Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host Microbe 1, 77–83 (2007).
Kuang, S. et al. Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis. Proc. Natl Acad. Sci. USA 114, 10642–10647 (2017).
Ruan, J., Xia, S., Liu, X., Lieberman, J. & Wu, H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature 557, 62–67 (2018).
Xia, S. et al. Gasdermin D pore structure reveals preferential release of mature interleukin-1. Nature 593, 607–611 (2021).
Kobayashi, T. et al. The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection. Cell Host Microbe 13, 570–583 (2013).
Chung, L. K. et al. The Yersinia virulence factor YopM hijacks host kinases to inhibit type III effector-triggered activation of the pyrin inflammasome. Cell Host Microbe 20, 296–306 (2016).
Ratner, D. et al. The Yersinia pestis effector YopM inhibits pyrin inflammasome activation. PLoS Pathog. 12, e1006035 (2016).
Li, Z. et al. Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11. Nature 599, 290–295 (2021).
Yin, H. et al. Insights into the GSDMB-mediated cellular lysis and its targeting by IpaH7.8. Nat. Commun. 14, 61 (2023).
Wang, Y. et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547, 99–103 (2017).
Zhang, Z. et al. Gasdermin E suppresses tumour growth by activating anti-tumour immunity. Nature 579, 415–420 (2020).
Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596583–589 (2021).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
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).
Acknowledgements
We thank the staff of beamline BL19U1 at the National Center for Protein Science (Shanghai) and the Shanghai Synchrotron Radiation Facility and beamline BL45XU at SPring-8 (Hyogo, Japan) for assistance with X-ray data collection, X. Ni (Shuimu BioSciences Co. Ltd) for assistance with cryo-EM data collection and the staff of the Sequencing Center and the EM Center at the National Institute of Biological Sciences, Beijing, for quantitative DNA sequencing and checking EM samples. This work was supported by the National Key R&D Program of China (2022YFA1304700), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-084), the CAS Strategic Priority Research Program (XDB29020202 and XDB37030202), the Basic Science Center Project (81788104) and Excellent Young Scholar Program (81922043) of NSFC, and a grant from the Youth Innovation Promotion Association of CAS. F.S. is also supported by the Tencent New Cornerstone Investigator Program.
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J.D. and F.S. conceived and supervised the study; X.Z. determined the crystal structures and performed biochemical assays; H.Z. reconstituted GSDMB pores for EM analyses and also performed in vitro liposome assays; Z.Z. contributed all the cellular data; Y. Su made the initial observation of isoform-dependent function of GSDMB; H.C. processed the cryo-EM data and generated the density map; Y.H. assisted H.Z. in cryo-EM data collection and built and refined the pore structure; Y. She, N.F. and J.W. provided technical assistance; and J.D. and F.S. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical characterization of IpaH7.8–GSDMB interaction.
a, Assay of GSDMB (or GSDMD) degradation by IpaH7.8. Flag-IpaH7.8 (WT or C357S) was co-expressed with GSDMB or GSDMD in 293T cells. Cell lysates were subjected to immunoblotting analyses. b, c, Co-immunoprecipitation assay of IpaH7.8 with human gasdermins (GSDMs). IpaH7.8C357S mutant (b) or IpaH7.8LRR (c) was co-expressed with an indicated Flag-EGFP-tagged GSDM in 293T cells. Cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting. d, f, Gel-filtration chromatography analyses of complex formation between IpaH7.8 or IpaH7.8LRR and GSDMB or GSDMD (FL or the C-terminal domain). Elution profiles along with SDS-PAGE analyses of complex elution fractions are shown. e, SPR profiles of IpaH7.8 binding to GSDMB or human or mouse GSDMD. g, h, Gel-filtration chromatography analyses of complex formation between IpaH7.8 (WT or indicated GSDMB-binding site mutants) and GSDMB (WT or indicated IpaH7.8-binding site mutants). The binding-site mutations in IpaH7.8 and GSDMB were identified from the complex crystal structure. All data are representative of three independent experiments. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 2 Structural and functional analyses of the autoinhibition of GSDMB.
a, b, Domain structural comparisons of GSDMB with other gasdermins. a, b, Cartoon structural models of the GSDMB-C domain (a) and the GSDMB-N domain (b) (second structural elements numbered) are compared with their counterparts in GSDMD (PDB: 6N9O) or mouse GSDMA3 (PDB: 5B5R). a, The linker regions between αG and αH in the three domains are highlighted. c, Mutagenesis analyses of the interdomain autoinhibition of GSDMB. GSDMB (WT or an indicated mutant to disrupt the autoinhibition) was transfected into 293T cells. Expression of the transfected GSDMB was examined by immunoblotting analyses. LDH release-based cell death data are means (bars) of three replicates (circles). d, Comparison of GSDMD and GSDMB liposome-leakage activity. Cardiolipin liposomes were treated with 0.6 or 0.3 µM purified GSDMBPP (isoform 3) or GSDMDPP in the presence or absence of PPase. Liposome leakage was monitored by measuring DPA chelating-induced fluorescence of the released Tb3+. Cleavage of GSDMBPP and GSDMDPP by PPase was analyzed by SDS-PAGE. e, Comparison of basal pyroptotic activity in FL GSDMB and GSDMD. Indicated amounts of GSDMB or GSDMD plasmid were transiently overexpressed in 293T cells. LDH release-based cell death data are means (bars) of three replicates (circles). Expression of the transfected constructs was examined by immunoblotting. Data (c–e) are representative of three independent experiments. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 3 Structural and functional analyses of IpaH7.8 recognition of GSDMD.
a, Structural modeling of IpaH7.8 recognition of GSDMD. Structure of GSDMD-N domain from FL GSDMD (PDB: 6N9O) is overlaid with that of GSDMB-N domain (from the IpaH7.8LRR–GSDMB complex). The binding interface is highlighted by a red box with structural elements labeled. b, Analyses of GSDMD degradation by IpaH7.8 mutants deficient in recognizing GSDMB. Flag-IpaH7.8 (WT, C357S, or a binding-site mutant) was co-expressed with GSDMD in 293T cells. Cell lysates were subjected to immunoblotting. c, d, Alignment of the N-terminal domain sequences of human gasdermins and mouse GSDMD. Second structural elements of GSDMB-N domain are numbered and shown on top of the sequences. Numbers of starting residues are indicated on the left. Identical residues are in red background and conserved ones are in red. Residues involved in binding IpaH7.8 are highlighted in cyan (c) or blue background (d). e, Functional validation of IpaH7.8 mutants in blocking GSDMD-mediated pyroptosis. Flag-IpaH7.8 (WT or an indicated mutant) and GSDMD were co-transfected into 293T cells and active CASP4 p20/p10 proteins were electroporated into the cells to induce GSDMD-dependent pyroptosis. IpaH7.8-induced degradation and CASP4 cleavage of GSDMD were examined by immunoblotting. SDS-PAGE analyses show the loading of CASP4 p20/p10 proteins. LDH release-based cell death data are means (bars) of three replicates (circles). f, Assay of mouse GSDMD degradation by IpaH7.8. Flag-IpaH7.8 (WT or C357S) was co-expressed in 293T cells with human or mouse GSDMD or human GSDMD with its L16−E21 sequences substituted with the mouse sequences (V16−D22). Cell lysates were subjected to immunoblotting. Data (b, e, f) are representative of three independent experiments. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 4 Sequence differences in GSDMB isoforms and characterizations of their functions.
a, Sequence alignment of GSDMB isoforms. Domain annotation is shown on top of the sequences. The exon 6 and 7 regions are labeled and highlighted by black boxes. GZMA cleavage site Lys244 (numbered after GSDMBiso3) is highlighted in blue background. The site for inserting a PPase-cleavage sequence is marked by a green arrow. Identical residues are in red background. Numbers of starting residues are indicated on the left. b, Gel-filtration chromatography analyses of complex formation between IpaH7.8 and different GSDMB isoforms, along with SDS-PAGE of the complex elution fractions. c, Assay of GSDMBIso3 in IpaH7.8 blocking of GZMA-induced pyroptosis. Flag-IpaH7.8 (WT or C357S) was transfected into 293T cells stably expressing GSDMBIso3; GZMA was electroporated into the cells. LDH release-based cell death data are means (bars) of three replicates (circles). Cell lysates were subjected to immunoblotting. d, 293T cells expressing different GSDMBPP isoforms were electroporated with PPase to cleave the GSDMBPP. Morphological examination of cell pyroptosis is shown. Scale bar, 25 μm. Data (b−d) are representative of three independent experiments. See Supplementary Fig. 1 for gel source data.
Extended Data Fig. 5 Isoform-dependent pore-forming activity of GSDMB.
a, Morphological assay of pyroptotic activities in different GSDMB isoforms. GSDMB-N domains derived from different isoforms were ectopically expressed in 293T cells. Scale bar, 25 μm. b, c, Cytotoxicity of GSDMB-N domains from different isoforms in bacteria. The N domains of indicated GSDMB isoforms under an IPTG-inducible promoter were transformed into E. coli. b, E. coli colonies on representative agar plates in the absence or presence of IPTG. c, Bacterial colony-forming units (CFU) per transformation of indicated isoforms are shown in the logarithmic form (log10), expressed as means (bars) of three replicates (circles). d–f, Direct CFU counting assay of the bacteriocidic activity in different GSDMB isoforms. The exponential-phase S. flexneri cultures were treated with the TBS media, purified PPase, or an indicated GSDMBPP isoform protein alone or being cleaved by the PPase. d, Serial dilution of the S. flexneri cultures on the agar plates. e, CFU counts of bacterial growth. f, SDS-PAGE analyses of PPase cleavage of the GSDMB isoform proteins. g–i, Liposome-leakage assays of pore-forming activity in GSDMB-N domains of different isoforms. Liposomes with indicated lipid compositions were treated with purified GSDMBPP in the presence or absence of PPase. g, Liposome leakage was monitored by measuring DPA chelating-induced fluorescence of the released Tb3+. h, Cleavage of GSDMBPP by PPase was analyzed by SDS-PAGE. i, Assay of the pore-forming activity on the control PE liposomes. All data are representative of three independent experiments.
Extended Data Fig. 6 Preparation of GSDMB pore samples and cryo-EM data processing.
a–d, Reconstitution, purification, and EM examination of GSDMBIso4 pores. a, Schematic diagram illustrating reconstitution and extraction of GSDMB pores from the liposomes. b, Purification of GSDMB pores by gel-filtration chromatography. The highlighted fraction containing homogenous pores were analyzed by SDS-PAGE and also applied to negative-stain EM examination and final cryo-EM data collection. Negative-stain EM image of GSDMB pores (representative of three independent experiments) is shown. Scale bar, 100 nm. c, Cryo-EM micrograph of GSDMB pores (representative of 7790 micrographs). Scale bar, 50 nm. d, Percentages of GSDMB pores with C26, C27, C28, C29 and C30-fold symmetry among a total of 270,000 top-view pore particles. e, Pore size distribution for GSDMBIso3. Inner diameters of a total of 8,600 GSDMBIso3 pores on the liposomes were measured. f, Processing of the cryo-EM dataset for GSDMB pore structure determination. Briefly, initial 3D model reconstruction generated a correct pore-like model. Iterative 2D and reference-guided 3D classifications were performed to select the best particles. A further round of 3D classification of the best particles indicates symmetry heterogeneity in the pores; a certain class of symmetry was chosen for final structural determination. Deep 2D classification allowed separation of the side view particles that were combined with best top view particles for 3D reconstruction and symmetry-imposed refinement to yield final cryo-EM maps.
Extended Data Fig. 7 Cryo-EM structural analysis of GSDMB pore.
a, Gold-standard Fourier shell correlation (FSC) plot for the 27-fold symmetric GSDMB pore. Horizontal dashed lines represent FSC cut-offs at 0.143. b, The cryo-EM density map of GSDMB pore is colored based on local resolution estimated using RESMAP in CryoSPARC. c, Validation of the cryo-EM density map. Close-up views show GSDMB subunit model fitted into the density map at five indicated locations.
Extended Data Fig. 8 Structural and functional analyses of GSDMB isoforms and their relevance to cancer cell pyroptosis.
a, Close-up view of structures around exon-6 region in the GSDMB-N domain. GSDMB-N structures in the autoinhibited state and in the pore are overlaid, with exon-6 regions highlighted in wheat. Relevant structural elements are labeled and crucial residues are in sticks. b, Superimposition of IpaH7.8LRR–GSDMBiso1 and IpaH7.8LRR–GSDMBiso4 complex structures. Close-up view shows the IpaH7.8–GSDMB binding interface in the two complexes and binding residues are in sticks. c, Sequence alignments of GSDMBIsoU and GSDMBiso4. Different residues are in magenta. Exon-6 sequences are in black background. GZMA cleavage site is in blue. Green arrow marks the site for inserting the PPase sequence. Identical residues are in red background. Numbers of starting residues are on top of the sequence. d, Cleavage of GSDMBIsoU by GZMA. WT or the cleavage-site lysine mutant of GSDMBIso3 or GSDMBIsoU proteins were incubated with GZMA, followed by SDS-PAGE analyses. e, Assay of pore-forming activity in GSDMBIsoU. Liposomes were treated with indicated isoforms of purified GSDMBPP in the presence of PPase. Liposome leakage was monitored by measuring DPA chelating-induced fluorescence of released Tb3+. SDS-PAGE analyses show cleavage of GSDMBPP. f, g, Cytotoxicity of the GSDMB-N domain of indicated isoforms. f, Indicated GSDMB-N domains were transfected into 293T cells and their expression was probed by immunoblotting. LDH release-based cell death data are means (bars) of three replicates (circles). g, Indicated GSDMB-N domains under an IPTG-inducible promoter were transformed into E. coli. CFUs per transformation are shown in the logarithmic form (log10) as means (bars) of three replicates (circles). h–j, Analyses of the pyroptosis-inducing function of GSDMBIsoU. H1299 cells stably expressing an indicated isoform were electroporated with a fixed dose (h, i) or titrating amounts of GZMA (j). h, Morphological examination of cell pyroptosis. Scale bar, 25 μm. i, j, Cleavage of GSDMB was probed by immunoblotting and ATP-based cell viability, expressed as means (bars) of three replicates (circles), is shown. k, l, Analyses of relative GSDMB isoform expression in primary epithelial cells derived from human gastrointestinal tissues. Indicated cells were left untreated (NT) or stimulated with IFN-γ, TNF-α, or LPS. k, GSDMB expression was examined by immunoblotting. l, Total mRNA transcripts of GSDMB were subjected to sequencing to quantify the different isoforms. Data are representative of three (d–j) or two (k, l) independent experiments. See Supplementary Fig. 1 for gel source data.
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Supplementary Fig. 1
This file contains the uncropped immunoblots for key data presented in the main text and Extended Data section of the manuscript.
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Zhong, X., Zeng, H., Zhou, Z. et al. Structural mechanisms for regulation of GSDMB pore-forming activity. Nature 616, 598–605 (2023). https://doi.org/10.1038/s41586-023-05872-5
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DOI: https://doi.org/10.1038/s41586-023-05872-5
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