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A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1

Abstract

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1,2,3,4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9,10,11,12, which stimulates the induction of type I interferons through the STING–TBK1–IRF-3 signalling axis13,14,15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17,18,19,20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNβ after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.

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Data availability

Atomic coordinates and structure factors of human TBK1 in complex with the human STING CTD EMW mutant, and mouse TBK1 in complex with the human STING CTT have been deposited in the Protein Data Bank (PDB) with accession codes 6O8B and 6O8C, respectively.

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Acknowledgements

The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health (NIH), National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source (ALS) is supported by the Director, Office of Science, Office of Basic Energy Sciences and US Department of Energy under contract number DE-AC02-05CH11231. We thank R. Vance and C. Kaplan for discussions. We acknowledge the use of electron microscopy facilities at the Biological Science Imaging Resource, which is supported by Florida State University and NIH grants S10 RR025080 and S10 OD018142. We acknowledge TAMU/LBMS for tandem mass spectrometry analyses. This research was supported in part by the Cancer Prevention and Research Institute of Texas (grant RP150454 to P.L.), the Welch Foundation (grant A-1931-20170325 to P.L.) and the NIH (grant R01 AI145287 to P.L. and R.O.W.).

Reviewer information

Nature thanks Andrea Ablasser, Philip J. Kranzusch and Osamu Nureki for their contribution to the peer review of this work.

Author information

P.L. conceived the study. B.Z. and F.D. expressed the proteins, conducted the binding studies and solved the structures. B.S. collected the data at ALS. B.Z., C.S., M.L., X.G. and Y.Lei. contributed to the cell-based studies. S.L.B. and R.O.W. generated the TBK1-knockout cells. A.P.W. and J.-Y.J. supervised some of the cell-based studies. P.X. conducted the cryo-EM studies. F.Z. and X.F. helped with electron microscopy data collection. Y.Liu., X.Z. and A.L. conducted tandem mass spectrometry analysis of phosphorylated STING. B.Z. and P.L. wrote the paper. A.P.W., R.O.W. and S.L.B. helped revise the manuscript.

Correspondence to Pingwei Li.

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Extended data figures and tables

Extended Data Fig. 1 Potential phosphorylation sites within the STING C-terminal tail.

a, List of phosphorylated STING peptides (residues 358–379, Ser358Trp) identified by LC–MS/MS. Phosphorylated residues are underlined and shown in orange. b, Representative MS/MS spectra of phosphorylated STING peptides (residues 358–379, Ser358Trp). b and c fragment ions are shown in red, and y and z fragment ions are shown in blue. Residues phosphorylated are shown in orange. Data are representative of two independent experiments. c, STING- and cGAMP-dependent activation of the IFNβ luciferase reporter in HEK293T cells. The cells were transfected with indicated amounts of the pcDNA3.1-hSTING plasmid (STING WT) and stimulated with cGAMP. Data are mean ± s.e.m. and representative of three independent experiments. Each dot represents a technical replicate (n = 3). ***P < 0.001, two-tailed Student’s t-test. d, Western blot of HEK293T cells transfected with the wild-type or ΔC9 mutant STING plasmid. Data are representative of three independent experiments. Source Data

Extended Data Fig. 2 SPR binding studies of human STING with TBK1.

a, Domain organization of human STING and truncated forms of STING used in this study. b, SDS–PAGE analyses of human STING (left) and human or mouse TBK1 (right) used in the SPR studies. cl, Top, SPR binding studies of human STING with human or mouse TBK1. Experiments without cGAMP in the running buffer are indicated. All others were conducted with 1 μM cGAMP. Bottom, the binding affinity (Kd) was determined by fitting the binding data to a one-site binding model. Data in bl are representative of at least two independent experiments. Source Data

Extended Data Fig. 3 Binding studies of human STING mutants with TBK1.

a, SDS–PAGE analysis of human STING mutants and human IRF-3 used in the SPR binding studies. bl, Top, SPR binding studies of human STING mutants (residues 155–379) with human TBK1 in the presence of 1 μM cGAMP. Bottom, the binding affinity (Kd) was determined by fitting the binding data to a one-site binding model. m, SPR binding study of the L374A STING mutant with IRF-3. n, The CTT of STING contains a highly conserved PLPLRT/SD motif. The sequence logo of STING is generated by WebLogo based on the sequence alignment of STING from mammals. The frequency of occurrence of an amino acid is indicated underneath the sequence. The PLPLRT/SD motif is downstream of the pLxIS motif that is involved in IRF-3 binding. o, SPR binding study of the high-affinity phosphomimetic EMW mutant of STING with human TBK1. p, IFNβ luciferase reporter assays of the S366A and L374A STING mutants. For each assay, HEK293T cells were transfected with pcDNA3.1-hSTING variants and stimulated with cGAMP. q, Time course IFNβ luciferase reporter assays of HEK293T cells transfected with wild-type and T376A mutant STING. The cells were stimulated with cGAMP. r, Western blot showing the phosphorylation of STING, TBK1 and IRF-3 in HEK293T cells transfected with wild-type or mutant (S366A or L374A) STING. Data in bm, o and r are representative of at least two independent experiments. Data in p and q are mean ± s.e.m. and representative of three independent experiments. Each dot represents a technical replicate (n = 3 in p and n = 6 in q). **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. NS, not significant. Source Data

Extended Data Fig. 4 Crystal structures of STING in complex with TBK1.

a, Ribbon representation of the structure of human TBK1 bound to the human STING CTD EMW mutant (residue 155–379, T376E, F378M and S379W). The kinase domains (KD) are in yellow and cyan, the ubiquitin-like domains (ULD) are in pink and red, the scaffold and dimerization domains (SDD) are in green and slate. The STING CTDs are shown by the blue and magenta ball-and-stick models. The TBK1 inhibitor BX795 is shown by the orange stick models. b, SDS–PAGE analysis of crystals of human TBK1 in complex with the human STING CTD EMW mutant. The data are representative of two independent experiments. c, Difference map of human STING CTT bound to mouse TBK1 contoured at 2.5σ. The σA-weighted Fo − Fc map was calculated with the STING CTT omitted from the model. The CTT of STING is shown by the slate ball-and-stick model. TBK1 dimer is shown by the ribbon representation coloured in green and cyan. d, Difference map of human STING CTD bound to human TBK1 contoured at 2.5σ. The σA-weighted Fo − Fc map was calculated with STING CTD omitted from the model. The CTT of STING is shown by the purple stick model. The TBK1 dimer is shown by the ribbons coloured green and cyan. e, Anomalous difference maps of Se-Met derivative of the human STING CTT EMW mutant bound to human TBK1. The blue map was calculated with model phases (ϕc) and the magenta map was calculated with experimental phases after density modification (ϕdm). The STING peptide is shown by the magenta ribbon and TBK1 shown by the green and cyan ribbons. f, Superposition of the structures of the human STING CTD EMW mutant (magenta) and human STING CTT (yellow) bound to human and mouse TBK1. Mouse TBK1 is shown by the green and cyan cartoon representation. Residues Glu376, Met378 and Trp379 from the STING CTD EMW mutant are shown by the magenta ball-and-stick models. Residues Thr376, Phe378 and Ser379 from the STING CTT are shown as the yellow ball-and-stick models.

Extended Data Fig. 5 Binding studies of human STING with human TBK1 mutants and characterization of TBK1-knockout HEK293T cells.

ag, SPR binding studies of the phosphorylated human STING CTD (residues 155–379) with human TBK1 mutants in the presence of 1 μM cGAMP. The binding affinity (Kd) was determined by fitting the binding data to a one-site binding model. SDS–PAGE analysis of proteins used in these studies is shown in the inset of panel a. h, Western blot characterization of TBK1-knockout HEK293T cell lines. i, IFNβ luciferase reporter assays using TBK1-knockout cells. For each assay, 0.2 ng pcDNA3.1-hSTING plasmids and/or 1.0 ng pcDNA3.1-hTBK1 plasmids were transfected into TBK1-knockout cells. Data are mean ± s.e.m. and representative of three independent experiments. Each dot represents a technical replicate (n = 3). P < 0.001, two-tailed Student’s t-test. j, Western blot showing the phosphorylation of TBK1, STING and IRF-3 in TBK1-knockout cells transfected with STING and TBK1 plasmids. TBK1-knockout cells were transfected with 0.2 ng pcDNA3.1-hSTING plasmids and/or 1.0 ng pcDNA3.1-hTBK1 plasmids and stimulated with cGAMP. k, Immunoprecipitation and immunoblot of Flag–STING and TBK1 in TBK1-knockout cells. The cells were transfected with Flag–STING and TBK1 mutants and stimulated with cGAMP. Flag–STING and TBK1 in the pull-downs and whole-cell lysates were analysed by immunoblotting. STING was visualized with Flag antibody. TBK1 in the pull-downs was detected with an antibody against TBK1. Data in ah are representative of at least two independent experiments. Western blot data in j and k are representative of three independent experiments. Source Data

Extended Data Fig. 6 cGAMP binding induces the oligomerization of full-length STING.

a, Gel-filtration chromatography analyses of full-length STING in the presence and absence of 1 μM cGAMP using a Superose 6 column. b, SDS–PAGE analyses of fractions containing full-length STING from gel filtration chromatography. c, Gel filtration chromatography analyses of full-length STING in 0.1% DDM or Amphipol A8-35 using a Superose 6 column in the presence of 1 μM cGAMP. d, SDS–PAGE analysis of cross-linked full-length STING. e, SEC-MALS analysis of full-length STING in 0.1% DDM and 1 μM cGAMP. f, Representative cryo-EM micrograph of full-length STING stabilized with Amphipol A8-35. g, Representative 2D averages of full-length STING particles in Amphipol A8-35. hj, Three views of 12 Å resolution map of STING oligomers. Human STING CTD dimers bound to cGAMP were docked into the map. k, A list of human STING mutants in the transmembrane domain. l, Gel-filtration chromatography analyses of wild-type and mutants of full-length STING in the presence of 1 μM cGAMP using a Superose 6 column. m, SDS–PAGE analyses of fractions of wild-type STING and STING mutants purified by gel filtration chromatography using a superose 6 column. n, IFNβ luciferase reporter assays showing that the mutations in the N-terminal transmembrane domain affect STING-mediated signalling. Indicated amounts of pcDNA3.1-hSTING plasmids were transfected into HEK293T cells. Data are mean ± s.e.m. and representative of three independent experiments. Each dot represents a technical replicate (n = 3). P < 0.001, two-tailed Student’s t-test. NS, not significant. o, Western blot showing that mutations in the transmembrane domain of STING affect the phosphorylation of STING, TBK1 and IRF-3. HEK293T cells were transfected with indicated amounts of pcDNA3.1-hSTING plasmids and stimulated with cGAMP. p, Confocal microscopy images of HEK293T cells transfected with wild-type STING and STING mutants with or without cGAMP stimulation. Scale bars, 20 μm. Data in ae, l and m are representative of at least two independent experiments. Data in f, g, o and p are representative of at least three independent experiments. Source Data

Extended Data Fig. 7 Proposed model for the recruitment and activation of TBK1 and IRF-3 through the cGAS-STING pathway.

(1) cGAS is activated by double-stranded DNA (dsDNA) in the cytosol and catalyses the synthesis of cGAMP from ATP and GTP. (2) cGAMP binding induces the oligomerization of STING at the ER or Golgi membranes. (3) TBK1 is recruited to the STING oligomers via its C-terminal PLPLRT/SD motif and activated by induced proximity in trans. Phosphorylation of STING by TBK1 increases the binding affinity between TBK1 and STING and facilitates further recruitment and activation of TBK1. (4) Activated TBK1 phosphorylates STING at the pLxIS motif, allowing it to recruit IRF-3 to the signalling complex. (5) The proximity of TBK1 and IRF-3 bound to adjacent STING molecules within the cGAMP–STING oligomer causes the phosphorylation of the pLxIS motif of IRF-3. (6) Phosphorylated IRF-3 dissociates from STING, oligomerizes, translocates to the nucleus, and initiates the transcription of IFNB gene.

Extended Data Table 1 Binding affinities of human STING mutants with TBK1
Extended Data Table 2 Sequences of STING C-terminal region from 60 mammals
Extended Data Table 3 Data collection and refinement statistics

Supplementary information

Supplementary Figure 1

This file contains the uncropped images with molecular mass markers for Western blot and SDS-PAGE.

Reporting Summary

Source data

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Fig. 1: The nine C-terminal residues of STING are crucial for STING-mediated signalling and binding of TBK1.
Fig. 2: A conserved PLPLRT/SD motif within the C-terminal tail of STING mediates TBK1 recruitment and activation.
Fig. 3: Crystal structures of STING in complex with TBK1.
Fig. 4: Mutations at the TBK1–STING interface affect STING binding and signalling.
Extended Data Fig. 1: Potential phosphorylation sites within the STING C-terminal tail.
Extended Data Fig. 2: SPR binding studies of human STING with TBK1.
Extended Data Fig. 3: Binding studies of human STING mutants with TBK1.
Extended Data Fig. 4: Crystal structures of STING in complex with TBK1.
Extended Data Fig. 5: Binding studies of human STING with human TBK1 mutants and characterization of TBK1-knockout HEK293T cells.
Extended Data Fig. 6: cGAMP binding induces the oligomerization of full-length STING.
Extended Data Fig. 7: Proposed model for the recruitment and activation of TBK1 and IRF-3 through the cGAS-STING pathway.

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