Gasdermin D pore structure reveals preferential release of mature interleukin-1

Abstract

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1β and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis^1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-1¹⁰. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1β and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1β upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Extended Data Fig. 1 |. Reconstitution and purification of GSDMD assemblies.

a, Optimized construct for human GSDMD referred to as “WT” GSDMD for convenience. The N-terminal MBP tag and the TEV-cleavable linker between MBP and GSDMD-NT are not shown. b, Schematic of GSDMD pore and prepore reconstitution. c, Reduced, but not abolished, activity of the GSDMD L192E mutant shown by Tb³⁺ leakage (n = 3 biological replicates). En: Activating enzyme. d, e, Size-exclusion chromatography profiles (d), and their locally enlarged views (e). The dashed box encloses the fractions containing the majority of GSDMD WT or L192E assemblies. The shaded fractions containing the least aggregated particles (e) were used for EM data collection. f, SDS-PAGE showing WT GSDMD-NT at around 30 kDa from the corresponding fractions in (e). g, Detergent screen. Non-ionic detergents known as ethylene glycol monoethers, with formula C_xE_y, yielded stable GSDMD pores. C₁₂E₈ was chosen as the final solubilizing agent. All scale bars: 200 nm. h, GSDMD pores extracted by 1% C₁₂E₈ from liposomes containing different types and amounts (%) of acidic lipids. Liposomes containing 20% PA were chosen. All scale bars: 200 nm. i, Sizes of GSDMD and GSDMA3 assemblies reconstituted on liposomes containing different types of acidic lipids (20%) and extracted by different types of detergents (1% C_xE_y, or 50 mM cholate), shown by outer diameters measured under negative-stain EM (from left to right, n = 64, 42, 77, 73, 14, 34, 45, 23, 21, and 18 particles). Data shown in c and i are mean ± s.d.. Data shown in f–h are representative of three independent experiments.

Extended Data Fig. 2 |. Cryo-EM data processing for the WT GSDMD dataset.

a, A cryo-EM image of the WT GSDMD sample. Scale bar: 100 nm. b, Processing of the WT GSDMD cryo-EM dataset. Initial 2D classes showed a ring-stacking phenomenon, which added to structural heterogeneity and posed challenges to symmetry determination. Density subtraction was therefore performed, followed by 3D reconstruction of each ring without assumption of symmetry, after which particle symmetry could be determined for certain 3D classes. These classes were then refined with their respective symmetry imposed to yield final cryo-EM maps. Data shown in a are representative of three independent experiments.

Extended Data Fig. 3 |. Cryo-EM data processing for the L192E GSDMD dataset.

a, A cryo-EM image of the L192E GSDMD sample. Scale bar: 100 nm. b, Processing of the L192E GSDMD cryo-EM dataset. The L192E dataset was first processed following the procedures for the WT dataset. Cryo-EM maps obtained from 3D refinement with symmetry imposed were further classified without alignment to remove heterogeneous particles. Then, the best 3D classes were refined again to improve resolutions. A 3D reconstruction at 7.3 Å was further improved by symmetry expansion, 3D classification without alignment, 3D local refinement, and per-particle CTF refinement to reach the final map at 3.9 Å resolution. c, Similarity of cryo-EM maps generated from the WT and L192E datasets. Due to the higher resolutions, maps from the L192E dataset were chosen for model building. Data shown in a are representative of three independent experiments.

Extended Data Fig. 4 |. Analysis of cryo-EM densities and models.

a, Half-map-to-half-map and model-to-map Fourier shell correlation (FSC) for the 33-fold symmetric GSDMD pore and prepore from the L192E dataset. Horizontal dashed lines represent FSC cut-offs at 0.5 and 0.143. b, c, Pore-form (b) and prepore-form (c) GSDMD subunits fitted into their respective cryo-EM density. Arrows indicate secondary structural elements specified by residue numbers. d, Close-up views of the pore-form GSDMD structure fitted into its cryo-EM density at six representative locations denoted by residue numbers.

Extended Data Fig. 5 |. β-hairpin extension and prepore-to-pore transition.

a, Comparison between auto-inhibited, prepore-form, and pore-form GSDMD. The auto-inhibited GSDMD-NT was obtained from the crystal structure of full-length GSDMD (PDB: 6N9O). The β4 strand and α4 helix are invisible in the crystal structure and were modelled based on the crystal structure of full-length GSDMA3 (PDB: 5B5R). b, Formation of β-hairpins (HPs). The β3-β4-β5 region constitute the first extension domain (ED1), which transforms into HP1 by refolding. The β7-α4-β8 region represents ED2 and becomes HP2. c, Sequence alignment of human and mouse GSDMD with secondary structures and key residues denoted. Blue highlight: Responsible for lipid binding, through either hydrophobic or charged interactions. Green: At inter-subunit interfaces. Underscore: Important for membrane insertion. d, Conserved rigid-body movement of the globular domain (“palm”) towards the membrane-distal direction during GSDM pore formation, shown by alignment of the GSDMA3 pore structure (PDB: 6CB8) and prepore model at their central axes.

Extended Data Fig. 6 |. Hydrophobic anchor and basic patches of GSDMs.

a, Effects of mutations in the hydrophobic anchor on GSDMD pore formation assessed by Tb³⁺ leakage from liposomes (n = 3 biological replicates). En: Activating enzyme. b, GSDM sequences aligned at the hydrophobic anchor and BPs. Blue highlight: Basic residues at BPs. Green highlight: Hydrophobic residues of the anchor. Dashes: Gaps. c, The GSDMA3 prepore model with the β1-β2 loop highlighted in green and BP1 shown in blue. A GSDMA3 prepore subunit is also shown in two orientations. d, A side view of pore-form GSDMA3 (PDB: 6CB8), with electrostatic surface shown around the β1-β2 loop. The anchor and BP2 are boxed in green. e, Impairment of the pore-forming ability of GSDMA3 by mutations in the hydrophobic anchor, shown by Tb³⁺ leakage (n = 3 biological replicates). Anchor: L47F48W49 mutated to E. f, A cryo-EM density blob that likely represents heads of phospholipids near BP3. Basic residues in BP3 point towards the blob. g, Effects of mutations in BP1 (R7R10R11 to E), BP2 (R42K43K51R53 to E), and BP3 (K204E or R174E) on GSDMD activity evaluated by Tb³⁺ leakage (n = 3 biological replicates). h, Importance of BP2 for GSDMA3 pore formation, shown by Tb³⁺ leakage (n = 3 biological replicates). BP2: R41K42R43K44 mutated to E. i, Exposure of the hydrophobic anchor and BP2 upon removal of the GSDMD-NT/CT inter-domain linker. Surface representations are shown for auto-inhibited GSDMD (PDB: 6N9O) with and without the inferred linker (cyan curve connecting Q241 and T284). Purple: GSDMD-NT. Black: GSDMD-CT. Green: Anchor-BP2 region. Yellow: Two ends of the linker. Data shown in a, e, g, and h are mean ± s.d..

Extended Data Fig. 7 |. GSDM acidic patches and their mutations.

a, Locations of APs shown by aligned GSDM sequences. Dots: Strings of omitted uncharged residues. Red highlight: Acidic residues. Blue: Basic residues. Of note, the basic residues near the APs may face the membrane (such as those in BP3) and therefore do not necessarily weaken the acidity of the pore conduit. b, Assessment of alanine mutations of GSDMD APs 1 through 4 by Tb³⁺ leakage (n = 3 biological replicates). c, Assessment of alanine mutations of GSDMA3 AP1 and AP2 by Tb³⁺ liposome leakage (n = 3 biological replicates). d, Negative-staining EM images of WT and AP-mutant GSDMD and GSDMA3 assemblies, solubilized from liposomes using C₁₂E₈ and cholate, respectively. All scale bars: 100 nm. e, Outer diameters of WT or AP-mutant GSDMD and GSDMA3 assemblies, measured under negative-staining EM (n = 50 particles per group). Data shown in b, c, and e are mean ± s.d.. Data shown in d are representative of three independent experiments.

Extended Data Fig. 8 |. Liposome experiments and electrostatics analysis.

a, Unlysed liposomes (25–75% PS) evident in minimal LDH release when GSDMD was added at a sub-lytic concentration (1x = 0.5 μM) (n = 3 biological replicates). En: Activating enzyme. b, Release of CyC, CRYGD, and OCM from liposomes permeabilized by GSDMD shown by immunoblotting. c, Similar rates of GSDMD pore formation on liposomes of various acidic lipid contents (25–75% PS), according to Tb³⁺ release (n = 3 biological replicates). d, Preferential IL-1β release from liposomes (50% and 75% PS) perforated by GSDMD shown by immunoblotting. e, Release of pro-IL-18 and IL-18 from liposomes permeabilized by GSDMD shown by immunoblotting. f, Unlysed liposomes evident in minimal LDH release when GSDMA3 was activated at a sub-lytic concentration (1x = 0.5 μM) (n = 3 biological replicates). g, Release of pro- and mature IL-1β from liposomes perforated by GSDMA3 shown by immunoblotting. h, Release rates of IL-1β cargoes through GSDMD pores shown by fitted hyperbolic functions. i, Initial release rates (lowercase r) extrapolated from h. j, Charge differences among the cargoes (Δq) and rate ratios (uppercase R). k, Plot of ln(R) versus Δq for estimating the electrostatic potential (E) of the GSDMD pore conduit. l, Unlysed liposomes shown by lack of release of encapsulated bulky FITC-labelled dextrans (2 MDa) when SLO or PFO was added at a sub-lytic concentration (1x = 0.1 μM) (n = 3 biological replicates). m, Similar release of pro- and mature IL-1β from liposomes permeabilized by PFO. n, Electrostatics surfaces of the modelled PFO pore conduit. Data shown in a, c, f, and l are mean ± s.d.. Data shown in b are representative of three, and data shown in d, e, g, and m two, independent experiments.

Extended Data Fig. 9 |. Macrophages experiments.

a, Comparable protein expression shown by immunoblotting. b, c, Similar sensitivity to pyroptosis and death evasion by glycine protection (b) or low-dose nigericin treatment (c), shown by LDH release (n = 3 biological replicates). d, Cleavage of engineered GSDMA3 chimera (A3chim) by caspase-1 (C1), shown by SDS-PAGE of proteolysis reactions using purified proteins. e, Comparable expression of A3chim (Flag-tagged) and its AP mutants in GSDMD-KO cells. AP1: 4 D/E to A. AP2: 2 D/E to A. f, g, Preferential release of mature IL-1β from glycine-protected living iBMDMs permeabilized by A3chim, shown by immunoblotting (f) and LDH release (n = 3 biological replicates) (g). h, i, IL-1β release from GSDMD-KO iBMDMs expressing with WT or AP-mutant A3chim under glycine protection, shown by immunoblotting (h) and LDH release (n = 3 biological replicates) (i). j-l, IL-1β release from living GSDMD-KO iBMDMs expressing A3chim stimulated by low-dose nigericin, characterized by immunoblotting (j), LDH release (n = 3 biological replicates) (k), and ELISA (n = 3 biological replicates) (l). m, n, IL-1β release from low-dose nigericin-stimulated GSDMD-KO iBMDMs expressing WT or AP-mutant A3chim, evaluated by immunoblotting (m), LDH release (n = 3 biological replicates) (n), and ELISA (n = 3 biological replicates) (o). Data shown in b, c, g, i, k, l, n, and o are mean ± s.d.. Data shown in a, d, e, f, h, j, and m are representative of two independent experiments.

Fig. 1 |. GSDMD architecture and conformational changes.

a, Ribbon diagram and dimensions of the 33-subunit human GSDMD pore structure fitted into its cryo-EM density. The pore features a large transmembrane β-barrel and a globular domain on the cytosolic side. b, Structure of the pore-form GSDMD subunit fitted into the cryo-EM density. The structure resembles a human left hand, with the globular domain as the “palm”, the α1 helix as the “thumb”, the membrane-inserted β-hairpins as the “fingers”, and the β1-β2 loop as the “wrist”. c, Flexible junction between the globular domain and the β-barrel, revealed by alignment of GSDMD and GSDMA3 pores at the β-barrel. The central axes are misaligned due to different pore sizes. The globular domain of the GSDMD pore is more membrane-distal than that of GSDMA3 by an approximate angle of 16°. d, Ribbon diagram and dimensions of the 33-subunit GSDMD prepore structure superimposed with its cryo-EM density. e, Prepore-to-pore transition of a GSDMD subunit. The two structures are aligned by their central axes and overlaid at the α1 helices. As the β-strands insert into the membrane, the globular domain rotates away from the membrane by approximately 38°.

Fig. 2 |. Membrane interaction by multiple contact sites.

a, Membrane docking by the β1-β2 loop, the most membrane-proximal feature of the prepore. The loop contains a hydrophobic anchor flanked by basic residues of BP2. The relative positions of BP1 and the anchor-BP2 region are displayed using an individual prepore subunit in two orientations. b, Pore-form GSDMD with the hydrophobic anchor and BP2 boxed in green and electrostatic surface shown locally. c, Locations and conservation of the three BPs in GSDMD, at α1, β1-β2 (excluding the hydrophobic tip), and β7-β8 regions, respectively. Continuous line: Conserved. Dashed line: Modestly conserved. d, e, Potential membrane distortion around GSDMD prepore (d) and pore (e). A subunit of each is enlarged to show the inferred local curvature. The contrasting curvatures indicate convexity-to-concavity membrane remodelling.

Fig. 3 |. Pore conduit and cargo transport.

a, Electrostatics surface (−1 to +1 kT/e) of the GSDMD pore, with the membrane-bound side formed by BPs and the solvent-exposed side formed by APs. The GSDMA3 pore conduit is similarly acidic. b, Negative electrostatic coverage of the GSDMD pore conduit. Modelled AP1 (5 D/E to A) and AP2 (2 D/E to A) pores have locally neutral conduits. c, d, Cartoons (c) and release of dextran cargoes from liposomes perforated by GSDMD, quantified by FITC fluorescence (n = 3 biological replicates) (d). e, f, Electrostatics surfaces of three protein cargoes (PDB: 2B4Z, 1H4A, 1TTX) (e) and their release from liposomes permeabilized by GSDMD, evaluated by immunoblot quantification (n = 3 biological replicates) (f). g, h, Basification of IL-1β and IL-18 through caspase-1-induced maturation (g) and AP’s of the precursors shown by aligned sequences (h). H: Human. M: Mouse. Dashes: Gaps. Dots: Strings of omitted residues. i, j, Release of pro- (WT and AP’-mutant) and mature and IL-1β from liposomes permeabilized by WT GSDMD (i) and the reciprocal experiments (j). ProAP1’: 8 D/E to K. ProAP2’: 11 D/E to K. k, Release of pro- and mature IL-1β from liposomes perforated by SLO. l, Electrostatics surface of the modelled SLO pore conduit. Data shown in d and f are mean ± s.d.. Data shown in i-k are representative of two independent experiments.

Fig. 4 |. Preferential IL-1β release from macrophages.

a, b, Preferential release of mature IL-1β from glycine-protected living iBMDMs permeabilized by GSDMD, shown by immunoblotting (a) and LDH release (n = 3 biological replicates) (b). UT: Untreated. N: Nigericin. G: Glycine. Uppercase H: High dose at 20 μM. Sup: Supernatant. WCL: Whole cell lysate. Pro: Precursor. Mat: Mature. c, Comparison of IL-1β release across GSDMD-KO iBMDMs expressing with WT or AP-mutant GSDMD, and across pro-IL-1β-KO iBMDMs expressing WT or AP’-mutant pro-IL-1β. EV: Empty vector, a mock transduction control. d-f, Release of mature IL-1β from living iBMDMs without glycine protection, characterized by immunoblotting (d), LDH release (n = 3 biological replicates) (e), and ELISA (n = 3 biological replicates) (f). Uppercase L: Low dose at 0.5 μM. g, h, Comparison of IL-1β release across GSDMD-KO iBMDMs expressing WT or AP-mutant GSDMD without glycine protection, evaluated by immunoblotting (g) and ELISA (n = 3 biological replicates) (h). i, j, Comparable leakage of pro-IL-1β and mature IL-1β from GSDMD-KO iBMDMs perforated by SLO and PFO (i) at cytotoxic and non-toxic concentrations shown by ATP-based cell death (n = 3 biological replicates) (j). Uppercase H: High dose at 625 nM. Uppercase L: Low dose at 0.16 nM. k, Schematic diagram for GSDMD pore formation and IL-1 release. The question mark indicates other possible assembly mechanisms. Data shown in b, e, f, h, and j are mean ± s.d.. Data shown in a and d are representative of three, and data shown in c, g, and i two, independent experiments.