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Cryo-EM structure of the gasdermin A3 membrane pore

Abstract

Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel β-barrel is formed by two β-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning β-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer.

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Acknowledgements

This work was supported by US NIH grant DP1HD087988 (H.W.), R01Al124491 (H.W.), R01AI123265 (J.L.) and Charles A. King Trust Postdoctoral Fellowship Program (J.R., X.L.). We thank the University of Massachusetts Cryo-EM Core Facility and NCI National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research for data collection, and D. Ni and M. Liao for discussions.

Reviewer information

Nature thanks H. Saibil, J. Whisstock and B. Zuber for their contribution to the peer review of this work.

Author information

J.R., H.W. and J.L. conceived the study. J.R., S.X. and H.W. designed the experiments and analysed the data. J.R. reconstituted the pores, performed cryo-EM experiments and refined the structure. J.R. and S.X. analysed structure-based mutants. J.R., H.W. and S.X. analysed the structure. H.W., J.R., S.X. and J.L. wrote the manuscript. X.L. provided advice and comments on the manuscript.

Correspondence to Hao Wu.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Reconstitution of GSDMA3 pores in vitro.

a, A general procedure for GSDMA3 pore reconstitution. b, Size-exclusion chromatography of GSDMA3 pores extracted from liposomes (top) and Coomassie blue-stained SDS–PAGE of the collected fractions. c, Representative negative-stain electron microscopy images of GSDMA3. d, Representative negative-stain electron microscopy images of human GSDMD pores with double-rimmed side views. e, f, Representative cryo-EM images of GSDMA3 pores with double-rimmed side views (e) and HgCl2-treated GSDMA3 pores (f). Scale bar, 50 nm (cf). All results were confirmed at least three times as technical replicates.

Extended Data Fig. 2 Cryo-EM analysis of double- and single-ring GSDMA3 pores.

ac, Gold-standard FSC plots from two half-reconstructions refined separately in RELION for the 27-fold double-ring pore map (a), the 27-fold single-ring pore map (b) and the 28-fold single-ring pore map (c). Model-to-map correlations are shown for the 27-fold single-ring pore (b). d, e, Local resolution estimation generated by ResMap on the two half maps separately refined in a gold-standard procedure in RELION. Local resolutions are colour-coded on the densities. Highest resolution is observed at the β-barrel domain for both the single-ring pore (d) and the membrane-inserted ring of the double-ring pore (e). The globular domains and the additional ring exhibit relatively low resolution.

Extended Data Fig. 3 Cryo-EM density validation.

Close-up views of GDSMA3 subunit model fitted into the cryo-EM density map at four locations denoted by residue numbers.

Extended Data Fig. 4 Aligned sequences in the N-terminal region among GSDMA3, human GSDMA, mouse GSDMD and human GSDMD.

Secondary structures are shown for both the auto-inhibited conformation1 and the pore conformation of GSDMA3. Dotted lines represent disordered regions. Residue numbers are shown as dots every ten residues above the alignment for GSDMA3 and below for human GSDMD. Blue highlights, potential positively charged residues for acidic lipid binding; blue, tested residues that did not affect pore formation; magenta and cyan highlights, residues involved in the oligomerization or membrane insertion of the two adjacent subunits; red and green highlights, previously reported and cancer-associated mutations localized on the oligomerization interface of the globular domains (green) and β-barrel domain (red).

Extended Data Fig. 5 Lipid binding by GSDM.

a, Effect of the GSDMA3 R132A/R145A mutation on the liposome-leakage activity, monitored by measuring DPA-chelating-induced fluorescence of released Tb3+ ion (n = 3 biological replicates). Error bars denote mean ± s.d. b, Equal processing of GSDMA3 by the 3C protease for the wild type and the α1 mutant. A representative gel of two independent experiments is shown.

Extended Data Fig. 6 Two-residue shift in the hydrogen-bonding pattern in each GSDMA3 subunit.

The shift results in a shear number of 27 × 2 = 54 for a 27-fold symmetric pore.

Extended Data Fig. 7 Structure-based sequence alignment.

The N-terminal region of GSDMA3 crystal structure (PDB ID: 5B5R) aligned with structures of perfringolysin O (5DIM), pneumolysin (4QQQ), haemolysin (3HVN), perforin-1 (3NSJ), complement C8 alpha (2QQH) and Plu-MAPCF (2QP2) and using the distance alignment matrix method (DALI). Helices are coloured in green, strands in cyan and loops in yellow. Vertical yellow bars indicate disordered regions in the GSDMA3-NT structure. Vertical red bars denote gaps in alignment of Plu-MACPF. Identical residues conserved among at least three proteins are highlighted in yellow. Glycines conserved among MACPF/CDCs are not conserved in GSDMA3, as highlighted in deep red.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Video 1

The GSDMA3-NT pore structure Rotational view of the 3.8 Å cryo-EM density (grey) superimposed with the atomic model (cyan) of the Hg-treated, 27-fold symmetric GSDMA3-NT pore.

Reporting Summary

Supplementary Video 2

Conformational change of GSDMA3-NT during pore formation The α3’, β3, β4, β5 region including disordered loops (ED1) in the autoinhibited structure1 extends into the first transmembrane β-hairpin (HP1) in the pore conformation. The β7, α4, β8 region including disordered loops (ED2) in the autoinhibited structure stretches to form the second transmembrane β-hairpin in the pore conformation (HP2).

Supplementary Video 3

Conformational change of pneumolysin during pore formation Long β-hairpins in pore-form pneumolysin (PDB: 5YL6)22 are formed by the straightening of a cluster of helices connected to the central β-sheet (PDB: 5CR6)23.

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Further reading

Fig. 1: Structure determination of GSDMA3 pores.
Fig. 2: Structure and conformational changes of the GSDMA3 pore.
Fig. 3: Mechanism of lipid recognition.
Fig. 4: Mechanism of oligomerization and membrane insertion.
Fig. 5: Comparison with MACPF/CDC proteins.
Fig. 6: A possible GSDMA3 pre-pore conformation.
Extended Data Fig. 1: Reconstitution of GSDMA3 pores in vitro.
Extended Data Fig. 2: Cryo-EM analysis of double- and single-ring GSDMA3 pores.
Extended Data Fig. 3: Cryo-EM density validation.
Extended Data Fig. 4: Aligned sequences in the N-terminal region among GSDMA3, human GSDMA, mouse GSDMD and human GSDMD.
Extended Data Fig. 5: Lipid binding by GSDM.
Extended Data Fig. 6: Two-residue shift in the hydrogen-bonding pattern in each GSDMA3 subunit.
Extended Data Fig. 7: Structure-based sequence alignment.

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