Activation of STING Based on Its Structural Features

Abstract

The cGAS-cGAMP-STING pathway is an important innate immune signaling cascade responsible for the sensing of abnormal cytosolic double-stranded DNA (dsDNA), which is a hallmark of infection or cancers. Recently, tremendous progress has been made in the understanding of the STING activation mechanism from various aspects. In this review, the molecular mechanism of activation of STING protein based on its structural features is briefly discussed. The underlying molecular mechanism of STING activation will enable us to develop novel therapeutics to treat STING-associated diseases and understand how STING has evolved to eliminate infection and maintain immune homeostasis in innate immunity.

Keywords: cyclic GMP-AMP; cyclic GMP-AMP Synthase (cGAS); cyclic dinucleotide; innate immunity; stimulator of interferon genes.

Figure 1

STING signaling pathway. After binding with the cGAMP (which is produced after the cGAS enzyme senses the double-stranded DNA), STING protein is activated followed by the activation of TBK1, which then phosphorylates transcription factor IRF3, which is then translocated into the nucleus of the cell leading to induction of interferon genes and ultimately anti-viral and anti-cancer immunity.

Figure 2

Domain organization of STING protein. (A) Schematic representation of human STING domain architecture. Blue bar: N-terminal domain; green bar: ligand binding domain (LBD); orange bar: C-terminal tail (CTT). DM, dimerization motif. PM, phosphorylation motif. TBM, TBK1 binding motif. (B) Sequence alignment of the region of LBDα1 from various species. The number is based on human STING. GXXXS motif is labeled as cyan bar.

Figure 3

Structures of LBD domain and full-length STING in apo and ligand-bound forms. (A) Structures of apo and antagonist bound LBD and apo full-length STING shown in cartoon. The secondary structures referred to in the text were labeled. The residues and compounds are shown as sticks. Gray rectangles represent TM domain. The black dashed square shows the magnified view of interactions between LBD and the N-terminal segment. The yellow dashed square shows the magnified view of LBD bound with antagonist C18. The connectors cross over each other. PDB IDs: 4F5W (human STING LBD), 4KC0 (mouse STING LBD), 6MXE (human STING LBD-C18), and 6NT5 (full-length human STING). (B) Structures of agonist bound LBD and full-length STING shown in cartoon. The binding of cGAMP induced the closed conformation and the 180° rotation of the LBD domain relative to the TM domain. The orange dashed square shows the magnified view of c-di-GMP (CDG) bound to STING LBD. The purple dashed square shows the magnified view of diABZI bound to LBD. The red dashed square shows the magnified view of cGAMP bound to LBD. The connectors are parallel to each other. The secondary structures referred to in the text were labeled. The residues and compounds were shown as stick. Gray rectangles represent the TM domain. PDB IDs: 4F5Y (human STING LBD-CDG), 6DXL (human STING LBD-diABZI), 4KSY (human STING LBD-cGAMP), and 6NT7 (full-length chicken STING–cGAMP).

Figure 4

Structural basis of ligand recognition and discrimination by STING. (A) Porcine STING LBD engages 2′3′-cGAMP (PDB ID: 6A06) with a partially open conformation (left panel). 2′3′-cGAMP adopts a fixed conformation in contrast to an alternative conformation that exists in other 2′3′-cGAMP-complexes. Superimposition of protomer A and protomer B (middle panel). The TP motif in two protomers, the key ligand recognition and discrimination element, adopts different conformations. The arrow denotes the tilt of the LBDα2–α3 helical bundle in protomer A relative to it in protomer B, 2Fo-Fc electron-density map for 2′3′-cGAMP (blue mesh) is shown and contoured at 2 σ (right panel). (B) Fly STING LBD-2′3′-cGAMP complex (PDB ID: 7MWZ) in a closed conformation (left panel). 2′3′-cGAMP adopts a fixed conformation in the complex structure. Superimposition of protomer A and protomer B (middle panel). The TP motif in two protomers adopts different conformations. The arrow denotes the tilt of the LBDα2–α3 helical bundle in protomer A relative to it in protomer B. The ligand discrimination residue N was shown. 2Fo-Fc electron-density map for 3′2′-cGAMP (blue mesh) and residue N is shown and contoured at 2 σ (right panel). Black dashed line shows the hydrogen bond.

Figure 5

Structural basis of STING signal transduction. (A) Crystal packing analysis shows that the ligand-bound LBD forms a linear array of STING dimer in a side-by-side manner in the crystal lattice. PDB ID: 4KSY. Gray square represents the TM domain. (B) Structural model of TBK1 binds to and phosphorylates STING CTT. Full-length chicken STING–cGAMP adopts a linear assembly with open ends. The cryo-EM density of the TBK1-CTT complex is shown. TBK1 binds to STING CTT (black dashed line) in a head-to-head manner and phosphorylates CTT from the adjacent STING dimer (CTT: green line). The zoomed-in view of the detailed interaction between TBK1 and CTT is shown (right panel). PDB ID: 6NT8 and 6NT9 for chicken STING–cGAMP tetramer and human TBK1-chicken STING CTT, respectively.

Figure 6

Structural basis of human STING oligomer induced by cGAMP and compound C53 which binds to a novel cryptic pocket in the TM domain (PDB ID: 7SII). (A) Cryo-EM density of the human STING/cGAMP/C53 oligomer shows a bent conformation. (B) Cartoon representation of the STING tetramer bound with cGAMP and C53 (left panel). The magnified black dashed square shows the interaction between two adjacent LBD domains (middle panel). The magnified orange dashed square shows the interaction network in the connector region. The residues referred to in the text were labeled. Black dashed line shows the hydrogen bond. (C) The oligomer interactions were observed in the TM domain (right panel). The detailed interaction between C53 and the STING TM domain (middle panel). The cryo-EM density for C53 contoured at 3 σ is shown in blue mesh. The black dashed line shows the hydrogen bond. (D) The localization of C88 and C91 in the oligomer structure. C88 is buried in the structure and C91 is located at TM3 and involved in the formation of the oligomer interface. C53 is shown in both sphere and stick modes.

Figure 7

Mapping SAVI mutations in the human STING structure (PDB ID: 6NT5). The SAVI mutations have been located on STING protein and divided into four clusters (1–4). Cluster 1 (black ball) is located at the N-terminal domain and includes the H72N variants. Cluster 2 (red ball) is localized at the dimer interface and shows the V147L/M, F153V, N154S, V155M, and G158A mutations. Cluster 3 (blue ball) is localized at the helical bundle of LBDα2–α3 and the long LBDβ1–β2 loop outside of the dimer interface and includes the C206Y/G, G207E, R281Q/W, and R284G/S mutations. Cluster 4 (purple ball) is located at the ligand-binding pocket and includes the G166E variant. Other than these clusters, there also exist combinations of two mutations; for example, S102P/F279L, V155E/L170Q, and L189V/S280R as shown by brown, green, and light brown balls, respectively.