Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA - PubMed (original) (raw)

. 2013 Aug 15;154(4):748-62.

doi: 10.1016/j.cell.2013.07.023. Epub 2013 Aug 1.

Manuel Ascano, Thomas Zillinger, Weiyi Wang, Peihong Dai, Artem A Serganov, Barbara L Gaffney, Stewart Shuman, Roger A Jones, Liang Deng, Gunther Hartmann, Winfried Barchet, Thomas Tuschl, Dinshaw J Patel

Affiliations

Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA

Pu Gao et al. Cell. 2013.

Abstract

Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.

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Figures

Figure 1

Figure 1. Crystal Structure of c[G(2’,5’)pA(3’,5’)p] Bound to hSTINGH232 and Details of Intermolecular Contacts in the Complex

(A) The 2.25 Å crystal structure of cyclic [G(2’,5’)pA(3’,5’)p] bound to hSTINGH232 (aa 155–341). The symmetrical hSTINGH232 dimer is shown in a ribbon representation, with individual monomers colored in magenta and yellow. α helices are labeled from α1 to α5. The c[G(2’,5’)pA(3’,5’)p] in a space-filling representation is bound in the central cavity at the interface between the two monomers. (B) An expanded view of the c[G(2’,5’)pA(3’,5’)p]-binding pocket in the complex. The position of H232 (in green) in a stick representation is labeled in this panel. (C) A surface representation of the structure of the complex shown in (A). (D) The view in (C) rotated through 90°. (E-G) Intermolecular contacts in the complex of c[G(2’,5’)pA(3’,5’)p] bound to hSTINGH232. The bound c[G(2’,5’)pA(3’,5’)p] is shown in biscuit color, with individual STING subunits in the symmetrical dimer shown in magenta and yellow. The bracketing of the purine rings of c[G(2’,5’)pA(3’,5’)p] by Y167 is shown in (E), and intermolecular contacts to the base edges and backbone phosphates of the ligand by the subunits of STING are shown in (F) and (G), respectively. Relates to Figure S1 and Table S1.

Figure 2

Figure 2. Comparison hSTINGH232 Complexes Bound to c[G(2′,5′)pA(3′ ,5′ )p] and c[di-GMP]

(A) Details related to alignment and hydrogen-bonding patterns within the four-stranded antiparallel β sheet that forms a cap over the binding pocket on formation of the c[G(2′,5′)pA(3′,5′)p]-hSTINGH232 complex. (B) Superposition of the c[G(2′,5′)pA(3′,5′ )p]-bound structure of hSTINGH232 (aa 155–341) with both subunits in green and c[di-GMP]-bound structure of hSTINGH232 (aa 139–379) with both subunits in beige (PDB: 4EF4). (C) Superposition of the c[G(2′,5′)pA(3′,5′)p] in green and c[di-GMP] in orange (PDB: 4EF4) in their complexes with hSTINGH232. (D) An expanded view in stereo of the top right segment of (B) following superposition of the c[G(2′,5′)pA(3′,5′)p]-bound structure of hSTINGH232 (both subunits in green) and c[di-GMP]-bound structure of hSTINGH232 (both subunits in orange) (PDB: 4EF4). Relates to Figure S1 and Table S1.

Figure 3

Figure 3. Crystal Structure of c[G(2′,5′)pA(3′,5′)p] Bound to mStingR231 and Comparison of Its Complex with the Same Ligand Bound to hSTINGH232

(A) The 1.77 Å crystal structure of c[G(2′,5′)pA(3′,5′)p] bound to mStingR231 (aa 154–340). (B) Intermolecular contacts to the cyclic dinucleotide ring system of the ligand by the subunits of mStingR231. (C) Superposition of the c[G(2′,5′)pA(3′,5′)p]-bound structures of hSTINGH232 (both subunits in green) and mSTINGR231 (both subunits in magenta). (D) Superposition of the c[G(2′,5′)pA(3′,5′)p] in its complexes with hSTINGH232 in green and mSTINGR231 in magenta. (E) The 1.9 Å crystal structure of c[G(2′,5′)pA(2′,5′)p] bound to hSTINGH232 (aa 155–341). (F) The 2.1 Å crystal structure of c[G(3′,5′)pA(3′,5′)p] bound to mStingR231 (aa 154–340). Relates to Figure S2 and Tables S1 and S2.

Figure 4

Figure 4. ITC Data on Binding of cGAMP Linkage Isomers to hSTINGH232 and Its Mutants, as well as to hSTINGR232, hSTINGA230/R232, mStingR231, and mStingA231

(A and B) ITC binding curves for complex formation between cGAMP linkage isomers bound to hSTINGH232 (aa 140–379) (A) and binding of c[G(2′,5′ )pA(3′,5′)p] to mutants of hSTINGH232 (B). (C) ITC binding curves for complex formation between cGAMP linkage isomers bound to hSTINGR232 (aa 140–379). (D) ITC binding curves for complex formation between cGAMP linkage isomers bound to hSTINGA230/R232 (aa 140–379). (E) ITC binding curves for complex formation between cGAMP linkage isomers bound to mStingR231 (aa 139–378). (F) ITC binding curves for complex formation between cGAMP linkage isomers bound to mStingA231 (aa 139–378). Relates to Tables S3 and S4.

Figure 5

Figure 5. Crystal Structure of DMXAA Ligand Bound to mStingR231

(A) Chemical formula of dimethylxanthenone-4-acetic acid (DMXAA). (B) The 2.4 Å crystal structure of two molecules of DMXAA bound to mStingR231 (aa 154–340). (C) Intermolecular contacts in the complex of DMXAA bound to mStingR231. The two bound DMXAA molecules are shown in biscuit color, with individual mSting subunits in the symmetrical dimer shown in magenta and yellow. The intermolecular contacts to the polar and nonpolar edges of the DMXAA by the mSting subunits are shown in two alternate views. (D) Superposition of the 2.4 Å DMXAA-bound structure of mStingR231 (both subunits in biscuit) and of the 1.77 Å c[G(2′,5′)pA(3′,5′)p]-bound structure of mStingR231 (both subunits in magenta). (E) Superposition of c[G(2′,5′)pA(3′,5′)p] in magenta and DMXAA in biscuit in their complexes with mStingR231. For dose dependence: Data points were determined in triplicate and are depicted as means ± SEM. Relates to Figure S3 and Table S2.

Figure 6

Figure 6. cGAMP Stimulation of the IFN Pathway in Mouse and Human Cells

(A) BMDMs (1 × 106) from C57B/6 mice were treated with increasing concentrations (5, 10, and 20 µM) of cGAMP linkage isomers, c[G(2′,5′)pA(2′,5′)p], c[G(2′,5′)pA(3′,5′ )p], and c[G(3′,5′)pA(3′ ,5′)p], and cells were collected at 4 hr after treatment. cGAMP linkage isomers were provided by addition into media. Mock treatment control was included. qPCR analyses of Ifnb1, Il6, and Ccl5 mRNAs were performed. (B) BMDMs from _Irf3_–/– and age-matched wild-type control mice were generated. Cells (1 × 106) were treated with cGAMP linkage isomers, c[G(2′,5′) pA(2′,5′)p], c[G(2′,5′)pA(3′,5′)p], and c[G(3′,5′) pA(3′,5′)p] at a final concentration of 15 µM. cGAMP linkage isomers were provided by addition into media. qPCR analysis of Ifnb1, Il6, and Ccl5 mRNAs were performed. (C) Murine BMDMs were incubated in media supplemented with indicated concentrations of cGAMP isomers for 18 hr (left) or for 30 min with and without Digitonin-mediated permeabilization (right). Eighteen hours later, IFN-α concentrations in the supernatant were determined by ELISA. (D) THP1 cells were incubated in media supplemented with indicated concentrations of cGAMP linkage isomers for 18 hr (left) or for 30 min with and without Digitonin-mediated permeabilization (right). CXCL10 concentrations were determined by ELISA after 18 hr. (E) Time course of STING-dependent IFN pathway activation by cGAMP linkage isomers. THP1 cells were incubated in media supplemented with cGAMP linkage isomers from 0 to 12 hr without permeabilization. IFNB1 and CXCL10 transcriptional activation was measured by RT-PCR, normalized against TUBA1B and vehicle control. Data points in (A) and (B) were determined in triplicate and are depicted as the mean ± SEM. A representative of two independently performed experiments is shown. For bar graphs, data points were determined in triplicate and are depicted as means ± SEM. Data points in (C) and (D) were determined in triplicate and are depicted as the mean ± SEM. Related to Figure S4 and Table S5.

Figure 7

Figure 7. Mouse and Human STING Mutational Analysis

(A) HEK293T cells were transfected with reporter constructs and human or murine STING expression plasmids as indicated. After 12 hr, cells were digitonin permeabilized to deliver cGAMP linkage isomers (5 µM concentration, 30 min permeabilization) and incubated for an additional 12 hr, followed by luciferase-reporter assay. (B) To gauge STING mutant stimulation by murine cGAS compared to the inactive cGAS mutant E211A, plasmids containing the indicated human or murine STING variants were cotransfected with either cGAS form and luciferase reporter constructs. Luciferase induction was determined after 30 hr. In this setting, the transfected plasmids provide the dsDNA stimulus for cGAS activation. Activation is expressed as fold induction in relation to control plasmid pMAX-GFP. (C) HEK293T cells were transfected as in (A) and stimulated with c[di-GMP](5 and 10 µM) following digitonin permeabilization. Luciferase activity was determined 12 hr after stimulation. As negative and positive controls, HEK293T cells transfected with hSTINGH232 were mock-treated (white bar) or stimulated with 5 µM c[G(2′,5′)pA(3′ ,5′)p] following digitonin permeabilization (green bar), respectively. (D) HEK293T cells were transfected as in (C) and after 12 hr stimulated with medium containing DMXAA (136 and 266 µM). Luciferase activity was measured after additional 12 hr. (E) ITC binding curves for complex formation between DMXAA and mStingR231. (F) ITC binding curves for complex formation between DMXAA and hSTINGR232/A162 and hSTINGR232/V162. (G) ITC binding curves for complex formation between DMXAA and hSTINGH232/A162 and hSTINGH232/V162. Data points in (A to D) were determined in triplicate and are depicted as the mean ± SEM. We have no explanation at this time for high-stoichiometry N values (∼1.5, instead of the expected 1) for the binding curves for A162 mutants of both hSTINGH232 and hSTINGR232 (F and G). Related to Figures S6 and S7, and Table S6.

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