Abstract
The stimulator of interferon genes (STING) innate immune pathway can exacerbate inflammatory diseases when aberrantly activated, emphasizing an unmet need for STING antagonists. However, no inhibitors have advanced to the clinic because it remains unclear which mechanistic step(s) of human STING activation are crucial for inhibition of downstream signaling. Here we report that C91 palmitoylation is not universally necessary for human STING signaling. Instead, evolutionarily-conserved C64 is basally palmitoylated and is crucial for preventing unproductive STING oligomerization. The effects of palmitoylation at C64 and C91 converge on the control of intradimer disulfide bond formation at C148. Together, dynamic equilibria of these cysteine post-translational modifications allow proper STING ligand-binding domain self-assembly and scaffolding function. Given this complex landscape, we took inspiration from STING’s natural autoinhibitory mechanism and identified an eight-amino-acid peptide that binds a defined pocket at the oligomerization interface, setting the stage for future therapeutic development.
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Data availability
Further information and requests for reagents should be directed to and will be fulfilled by the lead contact, L. Li (lingyin@arcinstitute.org). All source data can be found associated with this manuscript as follows: numerical data (source data), uncropped western blots (Supplementary Data 1–4), raw proteomic analysis results (Supplementary Data 5). Relevant structural data from the PDB were obtained from the following accession codes: 4F5W, 6NT5 and 7SII. Source data are provided with this paper.
References
Ablasser, A. & Hur, S. Regulation of cGAS- and RLR-mediated immunity to nucleic acids. Nat. Immunol. 21, 17–29 (2020).
Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP–AMP synthase. Cell 153, 1094–1107 (2013).
Li, X. et al. Cyclic GMP–AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity 39, 1019–1031 (2013).
Mankan, A. K. et al. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis. EMBO J. 33, 2937–2946 (2014).
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).
Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).
Zhang, C. et al. Structural basis of STING binding with and phosphorylation by TBK1. Nature 567, 394–398 (2019).
Zhao, B. et al. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins. Proc. Natl Acad. Sci. USA 113, E3403–E3412 (2016).
Decout, A., Katz, J. D., Venkatraman, S. & Ablasser, A. The cGAS–STING pathway as a therapeutic target in inflammatory diseases. Nat. Rev. Immunol. 21, 548–569 (2021).
Gulen, M. F. et al. cGAS–STING drives ageing-related inflammation and neurodegeneration. Nature 620, 374–380 (2023).
Ergun, S. L., Fernandez, D., Weiss, T. M. & Li, L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition. Cell 178, 290–301 (2019).
Zhang, X. et al. Cyclic GMP–AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013).
Hopfner, K.-P. & Hornung, V. Molecular mechanisms and cellular functions of cGAS–STING signalling. Nat. Rev. Mol. Cell Biol. 21, 501–521 (2020).
Liu, S. et al. The mechanism of STING autoinhibition and activation. Mol. Cell 83, 1502–1518 (2023).
Lu, D. et al. Activation of STING by targeting a pocket in the transmembrane domain. Nature 604, 557–562 (2022).
Shang, G., Zhang, C., Chen, Z. J., Bai, X. & Zhang, X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP–AMP. Nature 567, 389–393 (2019).
Xie, Z. et al. Structural insights into a shared mechanism of human STING activation by a potent agonist and an autoimmune disease-associated mutation. Cell Discov. 8, 133 (2022).
Haag, S. M. et al. Targeting STING with covalent small-molecule inhibitors. Nature 559, 269–273 (2018).
Humphries, F. et al. Targeting STING oligomerization with small-molecule inhibitors. Proc. Natl Acad. Sci. USA 120, e2305420120 (2023).
Jin, L., Lenz, L. L. & Cambier, J. C. Cellular reactive oxygen species inhibit MPYS induction of IFNβ. PLoS ONE 5, e15142 (2010).
Tao, L. et al. Reactive oxygen species oxidize STING and suppress interferon production. eLife 9, e57837 (2020).
Schmidt, B. & Hogg, P. J. Search for allosteric disulfide bonds in NMR structures. BMC Struct. Biol. 7, 49 (2007).
Dobbs, N. et al. STING activation by translocation from the ER is associated with infection and autoinflammatory disease. Cell Host Microbe 18, 157–168 (2015).
Ogawa, E., Mukai, K., Saito, K., Arai, H. & Taguchi, T. The binding of TBK1 to STING requires exocytic membrane traffic from the ER. Biochem. Biophys. Res. Commun. 503, 138–145 (2018).
Boncompain, G. et al. Synchronization of secretory protein traffic in populations of cells. Nat. Methods 9, 493–498 (2012).
Gao, X. & Hannoush, R. N. A decade of click chemistry in protein palmitoylation: impact on discovery and new biology. Cell Chem. Biol. 25, 236–246 (2018).
Mukai, K. et al. Activation of STING requires palmitoylation at the Golgi. Nat. Commun. 7, 11932 (2016).
Kemmoku, H. et al. Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol. Nat. Commun. 15, 220 (2024).
Vinogradova, E. V. et al. An activity-guided map of electrophile–cysteine interactions in primary human T cells. Cell 182, 1009–1026 (2020).
Barasa, L. et al. Development of LB244, an irreversible STING antagonist. J. Am. Chem. Soc. 145, 20273–20288 (2023).
Gui, X. et al. Autophagy induction via STING trafficking is a primordial function of the cGAS pathway. Nature 567, 262–266 (2019).
Liu, B. et al. Human STING is a proton channel. Science 381, 508–514 (2023).
Pryde, D. C. et al. The discovery of potent small molecule activators of human STING. Eur. J. Med. Chem. 209, 112869 (2021).
Yin, Q. et al. Cyclic di-GMP sensing via the innate immune signaling protein STING. Mol. Cell 46, 735–745 (2012).
Yu, X. et al. The STING phase-separator suppresses innate immune signalling. Nat. Cell Biol. 23, 330–340 (2021).
Mann, C. C. et al. Modular architecture of the STING C-terminal tail allows interferon and NF-κB signaling adaptation. Cell Rep. 27, 1165–1175 (2019).
Takahashi, M. et al. DrugMap: a quantitative pan-cancer analysis of cysteine ligandability. Cell 187, 2536–2556 (2024).
Njomen, E. et al. Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells. Nat. Chem. 16, 1592–1604 (2024).
Du, G. et al. ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D. Nature 630, 437–446 (2024).
Li, L. et al. Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–1048 (2014).
Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C. & Li, L. SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Mol. Cell 75, 372–381 (2019).
Acknowledgements
We thank A. Pawluk, B. Plosky, C. Ricci-Tam of the Arc Institute Scientific Publications Team for constructive feedback on the manuscript. We thank the P. Kim Lab at Stanford University for providing access to peptide synthesis equipment. We thank S. Pfeffer for helpful suggestions on using the RUSH system to study STING trafficking. We thank M. Cyert and J. Ferrell for their helpful discussion and suggestions on palmitoylation research and threshold setting, and all other Li Lab members for their constructive comments and discussion through the course of this study. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (award T32GM136631). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This research is funded by Stanford Medical School Dean’s startup fund and the Arc Institute.
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R.C. and X.C. conceived and coordinated the project, performed experiments, analyzed results and wrote the manuscript with input from all authors. S.L.E. conceived the project, performed experiments and analyzed results. E.N. performed the chemoproteomic experiments and analyzed the results. S.R.L. performed the NMR experiment and analyzed the results. C.R. analyzed the results of RNA-seq experiments. B.F.C. supervised E.N., provided scientific expertise, coordinated the project and approved the final draft of the manuscript. L.L. conceived the project, coordinated the project, wrote the manuscript and provided the project funding.
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Nature Chemical Biology thanks Wonsuk Chang, Hang Yin and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
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Supplementary Figs. 1–5 and Supplementary Tables 1 and 2.
Supplementary Data 1
Unprocessed blots for Supplementary Fig. 1.
Supplementary Data 2
Unprocessed blots for Supplementary Fig. 2.
Supplementary Data 3
Uncropped blots for Supplementary Fig. 3.
Supplementary Data 4
Uncropped blots for Supplementary Fig. 4.
Supplementary Data 5
Raw data from proteomic analysis.
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Source Data Fig. 1
Unprocessed western blots.
Source Data Fig. 2
Unprocessed western blots.
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 4
Unprocessed western blots.
Source Data Figs. 1–4
Statistical source data for Figs. 1–4.
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Chan, R., Cao, X., Ergun, S.L. et al. Cysteine allostery and autoinhibition govern human STING oligomer functionality. Nat Chem Biol 21, 1611–1620 (2025). https://doi.org/10.1038/s41589-025-01951-y
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DOI: https://doi.org/10.1038/s41589-025-01951-y
