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Regulation of STING activation by phosphoinositide and cholesterol

Nature (2026)Cite this article

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Abstract

Stimulator of interferon genes (STING) is an essential adaptor in the cytosolic DNA-sensing innate immune pathway1. STING is activated by cyclic GMP–AMP (cGAMP) produced by the DNA sensor cGAMP synthase (cGAS)2,3,4,5. cGAMP-induced high-order oligomerization and translocation of STING from the endoplasmic reticulum to the Golgi and post-Golgi vesicles are critical for STING activation6,7,8,9,10,11. Other studies have shown that phosphatidylinositol phosphates (PtdInsPs) and cholesterol also have important roles in STING activation, but the underlying mechanisms remain unclear12,13,14,15,16,17. Here we demonstrate that cGAMP-induced high-order oligomerization of STING is enhanced strongly by phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 and PtdIns(4,5)P2, and by PtdIns4P to a lesser extent. Our cryo-electron microscopy structures reveal that PtdInsPs together with cholesterol bind at the interface between STING dimers, directly promoting the high-order oligomerization. The structures also provide an explanation for the preference of the STING oligomer to different PtdInsPs. Mutational and biochemical analyses confirm the binding modes of PtdInsPs and cholesterol and their roles in STING activation. Our findings shed light on the regulatory mechanisms of STING mediated by specific lipids, which may underlie the role of intracellular trafficking in dictating STING signalling.

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Fig. 1: PtdInsPs enhance the high-order oligomerization of STING.
Fig. 2: The effect of PtdInsPs and cholesterol on STING phosphorylation by TBK1 on liposomes.
Fig. 3: The overall structures of the STING high-order oligomer bound to different PtdInsPs and agonists.
Fig. 4: PtdInsPs and CHS bind at the interface between two STING dimers.
Fig. 5: Mutational analyses of the binding interface in STING for PtdInsPs and cholesterol.

Data availability

The coordinates and cryo-EM maps of the structures reported in the study have been deposited in the RCSB and EMD databases, respectively, under the following accession codes: 9DAN and EMD-46694 (STING–cGAMP–STG2–PtdIns(4,5)P2), 9DAT and EMD-46697 (STING–cGAMP–STG2–PtdIns(3,5)P2), 9DAV and EMD-46699 (STING–cGAMP–STG2–PtdIns4P) and 9DAW and EMD-46700 (STING–cGAMP–C53–PtdIns(3,5)P2), EMD-71619 (STING–cGAMP–PtdIns(3,5)P2). No atomic model was built for the STING–cGAMP–PtdIns(3,5)P2 dataset owing to the low resolution of the map.

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Acknowledgements

Cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility, funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP220582. We thank D. Stoddard and J. Martinez Diaz for facility access; the members of the Structural Biology Laboratory at UTSW for equipment use; and M. Miletto for data collection. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work is supported in part by grants from the National Institutes of Health (R35GM150506 to J.X.T.; R01-AI093967 to Z.J.C.; R01CA273595 to X.Z. and X.-c.B.; and R01CA299257 to Z.J.C., X.Z. and X.-c.B.), the Welch foundation (I-1389 to Z.J.C., I-1702 to X.Z. and I-1944 to X.-c.B.) and the Cancer Grand Challenge (CGCFUL-2021\100007) with support from Cancer Research UK and the US National Cancer Institute (to Z.J.C.). X.-c.B. and X.Z. are Virginia Murchison Linthicum Scholars in Medical Research at UTSW. Z.J.C. is a Howard Hughes Medical Institute (HHMI) investigator.

Author information

Authors and Affiliations

  1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jie Li, Xuewu Zhang & Xiao-chen Bai

  2. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jay Xiaojun Tan & Zhijian J. Chen

  3. Center for Inflammation Research, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jay Xiaojun Tan & Zhijian J. Chen

  4. Aging Institute, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, USA

    Jay Xiaojun Tan

  5. Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Jay Xiaojun Tan

  6. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhijian J. Chen

  7. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xuewu Zhang

  8. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiao-chen Bai

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  1. Jie Li
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Contributions

X.-c.B. and X.Z. designed the study with input from J.L.; J.L. prepared protein samples for cryo-EM. X.Z. and X.-c.B. performed cryo-EM data acquisition and structure determination. J.L. performed the biochemistry experiments and cell-based assays. X.-c.B., X.Z. and J.L. analysed data. X.-c.B., X.Z., Z.J.C. and J.X.T. supervised the work and obtained funding. X.-c.B. and X.Z. wrote the initial draft. All of the authors contributed to the final draft.

Corresponding authors

Correspondence to Xuewu Zhang or Xiao-chen Bai.

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Nature thanks John Burke, Søren Paludan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 PIPs enhance the high-order oligomerization of STING to different extents.

(a) Analyses of the high-order oligomerization of STING treated with various concentration of PI(3,5)P2 and PI(4,5)P2 by native PAGE. The image shown here is a representative of three biological repeats. (b) Cryo-EM micrographs showing high-order oligomers of STING bound to different combinations of agonists and PIPs. cGAMP and STG2 induced relatively short oligomers of STING. Different PIPs promoted the formation of longer oligomers to different extents. The micrographs are representative images from more than 5,000 distinct regions. The scale bar is 500 Å.

Extended Data Fig. 2 Image processing procedure for the cryo-EM structure of STING bound to cGAMP/PI(3,5)P2/STG2 and the quality of the structure.

(a) Image processing procedure. (b) Gold-standard FSC curve of the final 3D reconstruction (left) and FSC between the map and atomic model (right). (c) Local resolution map of the final 3D reconstruction. (d) Sample densities of various parts of the structure.

Extended Data Fig. 3 Image processing procedure for the cryo-EM structure of STING bound to cGAMP/PI(4,5)P2/STG2 and the quality of the structure.

(a) Image processing procedure. (b) Gold-standard FSC curve of the final 3D reconstruction (left) and FSC between the map and atomic model (right). (c) Local resolution map of the final 3D reconstruction. (d) Sample densities of various parts of the structure.

Extended Data Fig. 4 Image processing procedure for the cryo-EM structure of STING bound to cGAMP/PI4P/STG2 and the quality of the structure.

(a) Image processing procedure. (b) Gold-standard FSC curve of the final 3D reconstruction (left) and FSC between the map and atomic model (right). (c) Local resolution map of the final 3D reconstruction. (d) Sample densities of various parts of the structure.

Extended Data Fig. 5 Comparison of the binding sites of different PIPs.

(a) Cryo-EM map and atomic model of the STING high-order oligomer in the presence of cGAMP, PI4P and STG2. PI4P and CHS have very weak density and therefore not included in the atomic model. (b) Comparison of the density of PIPs in the four structures presented in this study. The thresholds of the cryo-EM maps were set such that the sidechain of H16 is covered at similar levels in the four structures. It is clear that the density for PI4P is much weaker than PI(3,5)P2 and PI(4,5)P2 in the three other structures.

Extended Data Fig. 6 Image processing procedure for the cryo-EM structure of STING bound to cGAMP/PI(3,5)P2/C53 and the quality of the structure.

(a) Image processing procedure. (b) Gold-standard FSC curve of the final 3D reconstruction (left) and FSC between the map and atomic model (right). (c) Local resolution map of the final 3D reconstruction. (d) Sample densities of various parts of the structure. (e) Detailed view of the binding modes of PI(3,5)P2 and CHS at the interface between two STING dimers in the structure of STING bound to cGAMP/PI(3,5)P2/C53. (f) Cryo-EM density of PI(3,5)P2 and CHS in the structure of STING bound to cGAMP/PI(3,5)P2/C53.

Extended Data Fig. 7 Cryo-EM map and atomic model of STING oligomer in complex with cGAMP and PI(3,5)P2 determined in the absence of STG2 or C53

.

Extended Data Fig. 8 Lipid binding assay for purified STING.

(a) Binding of wild-type STING to a panel of lipids in a lipid-strip assay. (b) Binding of wild-type STING to an array of cholesterol and derivatives. (c) Comparison of binding to PIPs between wild-type STING and the K20E/R71H mutant. (d) Comparison of cholesterol binding between wild-type STING and the S80I mutant. The data shown are representative of three biological repeats.

Extended Data Fig. 9 Cholesterol may bind to STING without PIPs.

(a) The size exclusion chromatography profiles of wild-type STING and S80I mutant. The profile of the S80I mutant is similar to that of the wild type, suggesting that the S80I mutant is properly folded. (b) Cryo-EM map and atomic model of STING oligomer determined without PIPs (EMD-29282).

Extended Data Table 1 Cryo-EM data collection and structure refinement statistics
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 (uncropped gels for the Figures and Extended Data Figures).

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Li, J., Tan, J.X., Chen, Z.J. et al. Regulation of STING activation by phosphoinositide and cholesterol. Nature (2026). https://doi.org/10.1038/s41586-025-10076-0

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