Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING

Abstract

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.

Figure 1. Structure determination of the cGAS product

(A) Depiction of cGAMPs containing different combinations of phosphodiester bonds. (B) ¹H NMR spectra of the cGAS product and synthetic cGAMPs at 50°C in 30 mM NaH₂PO₄/Na₂HPO₄/D₂O buffer (pH 7.4). Substrate concentrations: natural cGAMP, 1.05 mM; 2′3′-cGAMP, 1.05 mM; 2′2′-cGAMP, 1.50 mM; 3′2′-cGAMP, 1.50 mM; 3′3′-cGAMP, 1.50 mM. (C) Tandem mass spectra of natural and synthetic cGAMPs resulting from higher energy collision dissociation (HCD) of the precursor ion ([M+H]⁺ = 675.107). (D) L929 and THP1 cells were transfected with HT-DNA and the cell extracts containing endogenous cGAMP were analyzed by tandem mass spectrometry along with the cGAS product and synthetic 2′3′-cGAMP. See also Figures S1 and S2.

Figure 2. cGAMP binding by STING as measured by ITC

(A) The original titration traces (top) and integrated data (bottom) of ITC experiments, in which c-di-GMP was titrated into a solution of STING dimer. (B and C) cGAS product (B) or synthetic 2′3′-cGAMP (C) was titrated into a solution of c-di-GMP bound STING, because the tight binding of 2′3′-cGAMP to STING made it difficult to do curve fitting if it were titrated into apo-STING dimer directly. (D) Summary of ligand binding affinities of STING for different cyclic di-nucleotides. For c-di-GMP, synthetic 2′2′- and 3′3′-cGAMP, the ligands were titrated into apo-STING dimer. For others, the ligands were titrated into c-di-GMP bound STING (3.55:1 in molar ratio). (E) Different concentrations of the cGAS product, synthetic cGAMPs and c-di-GMP were delivered into digitonin-permeabilized L929 cells. Four hours later, IFN-β RNA was measured by qRT-PCR. Dose response curves and the half maximal effective concentration (EC₅₀) for each compound were generated using GraphPad Prism 5.0 software. The error bars indicate standard deviations of triplicate experiments. See also Figure S3.

Figure 3. Ligand-induced conformational changes of STING revealed by the crystal structure of cGAMP-bound STING

(A) Overall structure of STING CTD bound to the cGAS product. Two perpendicular views are shown and the 2′3′-cGAMP molecule is indicated. STING forms a dimer and is colored in yellow and cyan for each molecule, respectively, in the right panel. The same color scheme is applied to other figures unless indicated otherwise. (B) Structural comparison of the apo- and cGAMP-bound forms of STING. The apo-STING dimer (PDB code: 4F9E, gray) is superimposed against one protomer of the cGAMP bound STING dimer. The arrow indicates the orientation of the conformational change in STING upon 2′3′-cGAMP binding. (C) Two new β sheets in each STING protomer are induced upon cGAMP binding. 2F_o-F_c electron density map, shown in blue mesh, is contoured at 1 σ on one chain. (D) The β sheets are stabilized through interdomain interactions. The residues that mediate interdomain interactions are shown in sticks. The distances between interacting atoms are in Å. All the structure figures are prepared in PyMol(DeLano, 2002). See also Figure S4.

Figure 4. A detailed view of the cGAS product within the STING structure

(A) Upper: the simulated annealing omit electron density map for cGAMP contoured at 3.0 σ. Lower: The 2F_o-F_c electron density map for cGAMP after refinement contoured at 1.0 σ. Two alternative conformations of cGAMP were built at 0.5 occupancy, respectively. (B) cGAMP, binding at a deeper pocket, drags the STING dimer (blue) closer to each other, compared to c-di-GMP bound STING (PDB code: 4F9G, gray). (C) The base groups of cGAMP are roughly parallel to each other and to four aromatic rings of the Tyr residues. (D) The bottom ribose ring (left panel) and the upper purine base groups (right panel) of cGAMP are coordinated by extensive polar contacts. Ser162 of STING interacts with 3′−OH of GMP. Only one conformation of 2′3′-cGAMP is shown for clarity. See also Figure S5.

Figure 5. Arg232 of human STING is essential for IFNβ induction by DNA and cGAMP

(A) L929 cells in which endogenous STING was stably knocked down by shRNA and replaced or poly(I-C) for 8 hr. IFN-β RNA levels were measured by qRT-PCR. (B) Extracts from the cell lines shown in (A) were immunoblotted with antibodies against STING or β-tubulin. (C) HEK293T cells stably expressing WT or mutant human STING-Flag were transfected with expression vectors for human cGAS or MAVS. 24 hr after transfection, IFN-β RNA levels were measured by qRT-PCR. (D) Extracts from the cell lines shown in (C) were immunoblotted with antibodies against STING or β-tubulin. The error bars indicate standard deviations of triplicate experiments.