Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP

Abstract

Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS)^1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain^9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP^9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses.

Extended Data Fig. 1 |. Flow chart of cryo-EM image processing for chicken STING in the apo state.

a, Representative micrograph. b, Representative 2D classes. c, Final reconstruction with colours based on local resolution. d, Gold-standard FSC curve of the final 3D reconstruction. e, Image processing procedure.

Extended Data Fig. 2 |. Flow chart of cryo-EM image processing for human STING in the apo state.

a, Representative micrograph. b, Representative 2D classes. c, Final reconstruction with colours based on local resolution. d, Gold-standard FSC curve of the final 3D reconstruction. e, Image processing procedure.

Extended Data Fig. 3 |. Structure of full-length chicken STING in the apo state.

a, Side view of the cryo-EM 3D reconstruction. The two subunits in the dimer are coloured in yellow and green. b, Cartoon representation of the structure in two orthogonal side views. c, Cartoon representation of the transmembrane domain dimer in the top view, from the cytosolic side. d, Interactions between the N-terminal segment and the body of chicken STING. e, Sequence alignment of STING from human, mouse and chicken (denoted by h-, m- and ch- prefixes, respectively). Secondary structure assignments are based on the structures. Residue numbers of human and chicken STING are shown above and below the aligned sequences, respectively.

Extended Data Fig. 4 |. Flow chart of cryo-EM image processing for cGAMP-bound chicken STING.

a, Representative micrograph. b, Representative 2D classes of the dimer (two top panels) and tetramer (bottom panel). c, e, Final reconstructions of the dimer and tetramer, respectively, coloured on the basis of local resolution. d, f, Gold-standard FSC curves of the final 3D reconstructions of the dimer and tetramer. g, Image processing procedure.

Extended Data Fig. 5 |. Additional mutational analyses of the transmembrane–LBD connector of human STING.

a, c, Effects of mutations in the connector and LBDα1 on cGAMP-stimulated IFNβ expression. Data are mean ± s.d. and representative of three biological replicates. b, d, Effects of STING mutations on phosphorylation of STING, TBK1 and IRF3. The result is representative of two independent experiments. The analyses for gene expression in a, c and phosphorylation in b, d were carried out in the same manner as in Fig. 2c and 2d, respectively.

Extended Data Fig. 6 |. Tetramer of full-length STING.

a, Rigid docking of the atomic model of the cGAMP-bound full-length chicken STING dimer (green and yellow) to the 3D reconstruction of the tetramer (grey). b, Left, tetrameric model of chicken STING in the apo state. The model is generated by superposition of two chicken STING inactive dimers on the active tetramer, on the basis of the transmembrane domain. Right, expanded view of the LBDα2–LBDα3 loop at the tetramer interface. The same loop in the active tetramer structure is shown for comparison; arrows indicate conformational differences between the two states. c, Model of full-length human STING tetramer. The model is constructed by superimposing two inactive human STING dimers on the active chicken STING tetramer, on the basis of the transmembrane domain. Right, top panel, packing between Phe153 in the connector loop and the LBDα2–LBDα3 loop. Right, bottom panel, Cys88 and Cys91—which have been shown to be palmitoylated—are highlighted. d, Cys206, Arg281 and Arg284—alterations of which cause constitutive activation of human STING—are located near the LBDα2–LBDα3 loop that forms the tetramer interface. These residues are highlighted in the tetramer model of the human STING LBD bound to cGAMP (based on PDB code 4KSY, as shown in Fig. 4c).

Extended Data Fig. 7 |. Sample density maps.

a–c, Sample density maps for various parts of chicken (a) and human (b) STING in the apo state, and cGAMP-bound chicken STING (c).

Extended Data Fig. 8 |. Data collection and model statistics.

a, Data collection and model refinement statistics. b, FSC curves between the maps and models.

Fig. 1 |. Structure of full-length human STING in the apo state.

a, Side view of the cryo-EM 3D reconstruction. The two subunits in the dimer are coloured in yellow and green. b, Cartoon representation of the structure in two orthogonal side views. c, Cartoon representation of the transmembrane domain dimer, viewed from the cytosolic side. d, Topological diagram of the transmembrane domain.

Fig. 2 |. Interaction between the N-terminal segment and the body of human STING.

a, Overview of STING N-terminal and body interaction in cartoon and surface representations. b, Detailed view of the interface. The view is expanded from the region in the box in a. c, Effects of STING mutation on the inter-domain interface in cGAMP-induced expression of the interferon-β 1 (IFNB1) gene. HEK293T cells that stably express IFNβ–luciferase reporter were transfected with expression vectors that encode wild-type (WT) or mutants of human STING. Cells were stimulated with three different concentrations of cGAMP (0, 0.3 and 1.4 μM). The R238A/Y240A mutant, which cannot bind cGAMP, was used as a negative control. Data are mean ± s.d. and representative of three biological replicates. d, Effects of STING mutations on the phosphorylation of STING, TBK1 and IRF3. HEK293T cells that express wild-type or mutant human STING were stimulated with cGAMP (1 μM) for 3 h and subjected to immunoblot analyses with indicated proteins. Data are representative of three biological replicates. e, Effects of STING mutations on the translocation and oligomerization of STING in cells. cGAS-deficient HeLa cells were transfected with expression vectors encoding wild-type or mutant STING–GFP. Representative confocal images with or without cGAMP stimulation are shown. Scale bar, 10 μm. The graph on the right is calculated from at least 150 cells. Data are mean ± s.d. and representative of three biological replicates.

Fig. 3 |. Structure of full-length chicken STING bound to cGAMP.

a, Side view of the 3D reconstruction. b, Cartoon representation of the structure. c, Comparison of the cGAMP-bound LBD in full-length STING with that of STING that lacks the transmembrane domain (PDB code 4KSY, shown in grey). d, The 180° rotation of the LBD relative to the transmembrane when converting from the inactive to the active state of STING. The connector and LBDα1, which are at the centre of the rotation, are highlighted. e, Effects of mutations in the connector and LBDα1 on IFNB1 gene expression, stimulated by three different concentrations of cGAMP (0, 0.3 and 1.4 μM). Data are mean ± s.d. f, Effects of STING mutations on phosphorylation of STING, TBK1 and IRF3. One representative set of results from three independent experiments is shown. g, Effects of STING mutations on STING translocation and oligomerization. Representative confocal images of STING–GFP with or without cGAMP stimulation are shown. Scale bar, 10 μm. Data are mean ± s.d. The analyses in e, f and g were conducted as in Fig. 2c, d and e, respectively. Data in e–g are representative of three biological replicates.

Fig. 4 |. Structure of STING tetramer.

a, Tetramer structure of cGAMP-bound chicken STING in the side and top views. The right panel shows an expanded view of the LBDα2–LBDα3 loop at the tetramer interface. b, Clashes at the LBDα2–LBDα3 loop in the tetramer model of inactive chicken STING, as constructed in Extended Data Fig. 6b. c, Tetramer of the human STING LBD bound to cGAMP. Two symmetry-related human STING LBD dimers in the structure (PDB code 4KSY) form the tetramer, which is similar to the chicken STING tetramer. d, Effects of mutations in the STING tetramer interface on cGAMP-induced formation of STING cellular puncta. Representative confocal images with or without cGAMP stimulation are shown. Scale bar, 10 μm. The experiments and analyses were conducted as in Fig. 2e. Data are mean ± s.d. and representative of three biological replicates. e, Cartoon model of cGAMP-induced conformational changes and oligomerization of STING.