2022 Liu Nature
2022 Liu Nature
2022 Liu Nature
https://doi.org/10.1038/s41586-022-05354-0 Ying Liu1,4, Pengbiao Xu1,4, Sophie Rivara1,4, Chong Liu1, Jonathan Ricci1, Xuefeng Ren2,
James H. Hurley2,3 & Andrea Ablasser1 ✉
Received: 17 February 2022
After activation and exit from the endoplasmic reticulum (ER), mechanism that explains how STING-dependent immune signalling
STING engages two bifurcating cellular effector pathways. The first is ended.
pathway diverges along STING’s transition to the Golgi and enables We tracked activated STING (phosphorylated at S366; hereafter pST-
autophagy, an ancestral antiviral function of STING; by contrast, ING)) (ref. 17) along its intracellular trafficking route by high-resolution
the second pathway, which begins at the Golgi, promotes the tran- confocal and Airyscan microscopy in HeLa cells. Extending previ-
scriptional activation of innate immune genes—an evolutionarily ous findings20,21, after stimulation with the small-molecule agonist
more recent functional adaptation22,23. Both pathways eventually diABZI-C3 (hereafter, diABZI) (ref. 3), pSTING rapidly (around 0.5 h)
converge at the lysosome, where STING is degraded21,23–25. The initia- appeared at the trans-Golgi network (TGN) and then moved to LAMP1+
tion of STING’s downstream transcription cascade is controlled by a endolysosomal compartments before disappearing (Extended Data
multi-step process: it begins with the recruitment of TBK1, continues Fig. 1a–d). pSTING transited RAB7+ late endosomes, but did not colocal-
with the phosphorylation of STING by TBK1 and results in the engage- ize with EEA1+, a marker of early endosomes (Extended Data Fig. 1c,d).
ment of interferon regulatory factor 3 (IRF3) by phosphorylated Membrane traffic between the TGN and endocytic organelles can
STING17,20,26,27. STING-bound IRF3, in turn, is phosphorylated by TBK1, involve clathrin-coated transport vesicles (CCVs)29. Of note, STING
and translocates to the nucleus to regulate gene expression jointly and pSTING were enriched in CCVs obtained from diABZI-stimulated
with NF-κB and other transcription factors. Through this cascade of cells, but not in those obtained from unstimulated cells (Fig. 1a).
molecular events, STING triggers a broad range of effector functions, Furthermore, pSTING colocalized with clathrin heavy chain, a defin-
most notably the expression of type I interferons (IFNs), proinflam- ing component of CCVs, at TGN46+ compartments in activated cells
matory cytokines and co-stimulatory molecules. Owing to these (Fig. 1b,c and Extended Data Fig. 1e). Stimulated emission deple-
favourable immunostimulatory properties, STING agonists are being tion (STED) super-resolution microscopy confirmed that pSTING is
developed for use as immunotherapeutic agents3,4,28. However, to incorporated into small (around 100-nm diameter) CCVs (Fig. 1d and
optimize robust immune activation while preventing immunopathol- Extended Data Fig. 2a). In correlated light and electron microscopy
ogy, STING responses require strict regulation. Here, we looked for a (CLEM) experiments, we observed that, upon activation, characteristic
Global Health Institute, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland. 2Department of Molecular and Cell Biology and California Institute for Quantitative
1
Biosciences, University of California, Berkeley, Berkeley, CA, USA. 3Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA. 4These authors contributed equally:
Ying Liu, Pengbiao Xu, Sophie Rivara. ✉e-mail: andrea.ablasser@epfl.ch
t
en
m
at
I
BZ
tre
A
o
(kDa)
di
N
50 Flag–
STING
100 pTBK1
50 pSTING CCV
15 AP-1σ
(pSTING on signal)
Region 1
STING
100 pTBK1
WCL
0.5
50 pSTING
Region 2
35 GAPDH
0
CHC
TGN46
e
f g 1,600
Fig. 1 | AP-1 loading of STING into CCVs at the TGN. a, Whole-cell lysate (WCL) microscopy (left) or electron microscopy at different Z-heights (three right
and CCV fractions from HeLa cells expressing Flag-tagged STING (HeLaSTING) panels). White arrows indicate CCVs. Scale bars, 0.5 µm. f, WCL and CCV
were analysed by western blot. Clathrin heavy chain (CHC) and GAPDH were fractions from HeLa cells that were treated with non-targeting control (NC)
used as loading and processing controls. b, Airyscan imaging of HeLaSTING cells small interfering RNA (siRNA) or AP-1 siRNA and stimulated with diABZI were
stimulated with diABZI. One representative cell of n = 7 cells. White arrows analysed by western blot. CHC was used as a loading control. g, Quantification
point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified of the area of pSTING in bright-field fluorescent microscopy images of
panels). c, Quantification of the colocalization of pSTING with CHC described HeLaSTING cells that were treated with siRNAs and stimulated with diABZI.
in b, using Manders’ colocalization coefficients. Mean ± s.e.m. of n = 7 cells. Mean ± s.e.m. of n = 3 independent experiments with 99 fields of view per
d, STED images showing pSTING enclosed in CCVs from cells transfected with condition. h, HeLa cells transfected with siRNAs and stimulated with diABZI
mCherry–clathrin and Flag–STING and stimulated with diABZI. Regions 1 and 2 were analysed by western blot. Ratios of STING versus loading control (GAPDH)
are magnified from a large-field-of-view STED image (see Extended Data normalized to the 0-h time point of each condition. One representative
Fig. 2a). Scale bars, 200 nm. e, CLEM of HeLaGFP–STING cells stimulated with example of three (a–c,h) or two (f) independent experiments is shown.
diABZI. The images depict box 3 of Extended Data Fig. 2b in Airyscan
STING perinuclear foci localized to areas that contain multiple CCVs in a marked reduction in the levels of STING, pSTING and pTBK1 within
(Fig. 1e and Extended Data Fig. 2b,c). Together, these results show that CCVs, whereas the overall cellular levels of STING and pSTING were
CCVs can function as transport vehicles of activated STING. increased (Fig. 1f–h and Extended Data Fig. 3a). Crucially, STING traf-
ficking from the ER to the TGN was not affected in AP-1-depleted cells
(Extended Data Fig. 3b). We confirmed that a knockdown of the σ1 subu-
AP-1 sorts STING into CCVs nit alone or a genetic knockout of the µ1 subunit in mouse embryonic
The formation of CCVs relies on heterotetrameric adaptor protein fibroblasts (MEFs) affected the degradation of STING, and that rein-
complexes, which physically connect clathrin with transmembrane troducing the missing subunit restored the decay of activated STING
cargo proteins29–31. The most prominent adaptor protein complex that (refs. 33,34) (Extended Data Fig. 3c,d). These findings suggest that AP-1
is involved in cargo shuttling from the TGN to endolysosomes is AP-1, has a role in controlling activated STING by gating its loading into CCVs.
which consists of the four subunits β1, γ, µ1 and σ1 (ref. 32). Notably, a Adaptor-protein-dependent sorting requires direct engage-
knockdown of three AP-1 subunits (AP1G1, AP1S1 and AP1B1) resulted ment between the multimeric complex and its cargo on target
(pSTING on signal)
0.5
TGN46
AP-1
b diABZI treatment (h) d
siNC siAP-1
(kDa) 0 1 2 4 6 7
diABZI (h) 0 0.5 1.0 1.5 2.0 4.0 0 0.5 1.0 1.5 2.0 4.0
15 AP-1σ
(kDa) 90 pTBK1
110 AP-1γ IP: Flag
Ratio: 1.0 1.7 1.1 1.0 0.2 2.1 2.8 2.6 2.6 2.0
50 Flag–
90 TBK1
STING
60 pIRF3 60 pIRF3
Ratio: 1.0 1.5 0.8 0.5 0.3 3.2 4.5 4.1 4.0 1.8
15 AP-1σ
WCL 60 IRF3
110 AP-1γ
40 pSTING
50 Flag–
STING Ratio: 1.0 1.1 0.9 0.8 0.6 2.5 2.7 3.2 5.8 3.8
40 pSTING
c
H ZI ent
STING
P
AM
AB tm
Ratio: 1.0 0.8 0.5 0.4 0.4 0.3 1.0 0.8 0.7 0.6 0.7 0.7
G
di rea
90 -1
2′ er
-c
SV
m
t
3′
o
N
AP-1γ
110
WCL f
Flag– siNC siAP-1
40 STING diABZI (h) 0 2 4 0 2 4
e P = 0.0003
(kDa) 90 pTBK1
P < 0.0001 200 P < 0.0001 120 2,000 P < 0.0001
250
IFNB1 (fold change)
60 pIRF3
IFIT1 (fold change)
200 100
150 1,500
80 40 pSTING
150
100 60 1,000
100 30 STING
40
50 500 Ratio: 1.0 0.8 0.3 1.0 0.9 0.5
50 20
0 0 0 0 15 AP-1σ
diABZI: – + – + diABZI: – + – + diABZI: – + – + diABZI: – + – + 40
GAPDH
siNC siAP-1 siNC siAP-1 siNC siAP-1 siNC siAP-1
Fig. 2 | AP-1 binding directs STING degradation to limit immune activation. diABZI and analysed by western blot. Vinculin was used as a loading control.
a, Airyscan imaging of HeLaSTING cells that were stimulated for 2.5 h with diABZI. e, Induction of IFNB1, IFIT1, IFIT2 and IFIT3 expression was assessed by
The colocalization of pSTING with AP-1γ is quantified by Manders’ quantitative PCR with reverse transcription (RT–qPCR) in HeLa cells
colocalization coefficients. One representative cell is shown, and the transfected with siRNAs and treated with diABZI for 3 h. Ratios of IFNB1, IFIT1,
quantification is the mean ± s.e.m. of n = 7 cells from one out of four IFIT2 and IFIT3 mRNA versus GAPDH mRNA normalized to the untreated groups
independent experiments. White arrows point at occurrences of pSTING. Scale of each condition. Data are mean ± s.d. of three technical replicates. P values
bars, 4 µm (left) or 1 µm (magnified panels). b, HeLaSTING cells were stimulated were obtained by two-tailed Student’s t-test. f, WI-38 human fibroblasts
with diABZI. After immunoprecipitation (IP) with anti-Flag antibody, cells were transfected with siRNAs for three days were stimulated with diABZI and
analysed by western blot. c, HeLaSTING cells were stimulated with diABZI for 2 h, analysed by western blot. GAPDH was used as a loading control. One
infected with HSV-1 (multiplicity of infection (MOI) = 10) for 6 h or transfected representative example of three (a,d–f) or two (b,c) independent experiments
with 90mer dsDNA (1 µg) for 3 h or 1 µM 2’3’-cGAMP for 6 h. After is shown. Ratios of target proteins versus loading control normalized to the 0-h
immunoprecipitation with anti-Flag antibody, samples were analysed by time point of each condition (d,f).
western blot. d, HeLa cells transfected with siRNAs were stimulated with
membranes30,31. Accordingly, pSTING colocalized with AP-1 at the of TBK1 in AP-1-depleted cells, consistent with a model in which
TGN, and STING robustly bound the γ and σ1 subunits of AP-1 after AP-1 functions as the initiator of signalling shutdown upstream of
activation (Fig. 2a,b and Extended Data Fig. 4a). STING associated lysosomes (Extended Data Fig. 4b). Cells that were depleted of AP-1
with AP-1 in response to diverse cellular activators of STING, includ- exhibited an increased type I IFN response, which correlated with a
ing double-stranded DNA (dsDNA), cyclic GMP-AMP (cGAMP) and more potent inhibition of HSV-1 replication (Fig. 2e and Extended
the physiological activator herpes simplex virus 1 (HSV-1), providing Data Fig. 4c). By contrast, suppression of AP-1 mildly decreased type
evidence that AP-1 recognition is an integral element of the cell bio- I IFN responses elicited by triphosphate RNA, a trigger for RIG-I-like
logical regulation of STING (Fig. 2c). helicases, showing that AP-1-mediated negative regulation of innate
immune activation is a specific feature of STING signalling (Extended
Data Fig. 4e). Analysis of various cells, stimuli and read-outs as well
AP-1 restricts STING signalling as single subunit knockdowns showed that AP-1-dependent restric-
We next determined how the disruption of AP-1 affects tion of type I IFN signalling is a universal mechanism for controlling
STING-dependent immune signalling. A knockdown of AP-1 increased the activity of STING (Fig. 2f and Extended Data Figs. 4f–i and 5a–d).
the levels of pTBK1 and pIRF3, in addition to pSTING, and prolonged Stronger STING-dependent type I IFN responses were also appar-
signalling when compared to stimulated control cells (Fig. 2d). ent in µ1A-deficient MEFs as compared to their µ1A-reconstituted
Blocking lysosomal acidification further boosted the activation counterparts (Extended Data Fig. 4j,k). In STING-associated
ΔCTT
T
LI
LI
W
W
WT
ELI
diABZI – – + +
LI
(kDa)
E
1 134 157 336 379
110 AP-1γ (kDa) 100 pTBK1
MSQEPELLISGMEKPLPLRTDFS 50 Flag–
IP: Flag
Flag–
CCV
50 STING
IRF3 STING
AP-1 TBK1
180 CHC
110 AP-1γ
WCL 100 pTBK1
50 Flag–
STING
50 Flag– WCL
STING
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
N C
120 CHC
d GST AP-1 + – – – + + – + + – + + – + + – e
ARF1 – + – – + – + + – + + – + + – + siNC siTBK1 siIRF3
STING WT – – + – + + + – – – + + + – – – diABZI – + – + – +
STING ELI – – – + – – – + + + – – – + + +
(kDa) (kDa) (kDa) 100 AP-1γ
Flag– IP: Flag
180 180 40
130 130 STING
GST–AP-1γ 100 100
His–AP-1β 70 70 100 TBK1
AP-1μ 55 55
SUMO STING 40 40 60 IRF3
GST tag 35 35
25 25 100 AP-1γ WCL
ARF1
AP-1σ Flag–
15 15
40
10 10 STING
40 GAPDH
M Individual M Input M Pull-down
protein
150 nM
diABZI (h) 0 1 2 4 0 1 2 4 0 1 2 4 0.20 75 nM 0.20
(kDa) 100 pTBK1 0.15 0.15
0.10 0.10
100 TBK1 Kd = 393 nM
0.05 0.05
55 pIRF3 0 0
0 300 600 900 0 200 400 600
IRF3 Time (s) Concentration (nM)
55 0.4 0.4
pSTING response (nm)
0.2 0.2
40 STING 600 nM
300 nM
0.1 150 nM 0.1 Kd = 63 nM
Ratio: 1.0 0.6 0.7 0.4 1.0 1.1 1.0 1.0 1.0 0.7 0.7 0.5 75 nM
0 0
120 Vinculin
0 300 600 900 0 200 400 600
Time (s) Concentration (nM)
Fig. 3 | TBK1-dependent phosphorylation controls the binding of STING to NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI.
AP-1. a, Schematic diagram of the CTT of human STING (hSTING) and sequence After immunoprecipitation with anti-Flag antibody, samples were analysed by
logo of the CTT as indicated from 50 species. b, HeLa STING-knockout (KO) western blot. GAPDH was used as a loading control. f, HeLa wild-type cells,
cells transfected with Flag-tagged STINGΔCΤΤ (Δ1–341), wild-type (WT) STING, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or
STINGE(E360A), STINGLI(L364A/I365A) or STINGELI(E360A/L364A/I365A) were 4 h were analysed by western blot. Ratios of target proteins versus loading
treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, control normalized to the 0-h time point of each condition. Vinculin was used
samples were analysed by western blot. c, WCL and extracted CCV fractions as a loading control. g, Bio-layer interferometry binding studies of LBD-STING
from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD.
STINGLI(L364A/I365A) and treated or not with diABZI were analysed by western The right graphs show the binding affinity of STING (top) and pSTING
blot. CHC was used as a loading control. d, Glutathione sepharose pull-down (bottom). One representative example of at least three (b,d,f) or two (c,e,g)
assays of wild-type LBD-STING or LBD-STINGELI by glutathione S-transferase independent experiments is shown.
(GST)-tagged AP-1 core with or without ARF1. e, HeLaSTING cells transfected with
AP-1σ
AP-1–ARF1 AP-1β
STING
ARF1
c ARF1 d e
AP-1σ E360
G367
pSTING tail E362
Q359 H85
AP-1β pS366
N
C I365
L364
ARF1
f g h Flag–STING
A
66
T
HA–AP-1σ
LR
S3
W
LI
(kDa)
Flag–
8D
V8 S
40 STING
03
T
KR
W
I1
(kDa) CCV
20 AP-1σ
15 HA
pS366
IP: Flag 180 CHC
Flag–
50
STING
Flag–
40 STING
R61 K60 15 HA
WCL 20 AP-1σ WCL
50 Flag–
STING 40 GAPDH
i j Clathrin–AP-1–STING
6A
A
66
cluster formation
W /36
/3
Clathrin
S3 A
S3 A
S3 A
S3 A
58
66
58
58
66
58
T
T
S3
S3
W
AP-1
diABZI – – – – + + + + IRF3
(kDa) 110 AP-1γ T
CTT CCVs
Lysosomal
50 Flag– IP: Flag G
STING degradation
STING
Fig. 4 | Structural basis for phospho-regulation of STING recognition by R61A), σ(I103S) and σ(V88D) were stimulated with diABZI for 2 h. Cell lysates
AP-1. a, Three-dimensional (3D) reconstructions of the complex in two different were extracted, immunoprecipitated with anti-Flag antibody and analysed by
orientations. The atomic models of dimeric human LBD-STING (Protein Data western blot. h, WCL and CCV fractions from untreated and diABZI-treated
Bank (PDB) code: 4KSY) and the AP-1 complex (PDB 6DFF) were fitted into the HeLa STING KO cells reconstituted with Flag-tagged wild-type STING and the
maps (grey) through rigid-body docking. b, High-resolution 3D reconstruction indicated STING mutants STINGLI, STINGLR(L374A/R375A) or STING(S358A)
from focused refinement on the AP-1 core and pSTING tail at 2.34-Å resolution were analysed by western blot. CHC and GAPDH were used as loading controls.
contoured at 3σ. c, Ribbon representation of the AP-1 pSTING complex i, HeLa STING KO cells reconstituted with Flag-tagged STING and the indicated
structure. d–f, Detailed views of the binding interface. e, Potentialhydrophobic STING mutants STING(S358A), STING(S366A) or STING(S358/366A) were
interactions around the EXXXLI motif are indicated with dotted lines. The stimulated with diABZI for 2 h or left untreated. After immunoprecipitation
density map (grey mesh) of pSTING tail is contoured at 3σ. f, Potential hydrogen with anti-Flag antibody, samples were analysed by western blot. j, Schematic
bonds around pS366 indicated with dotted lines. The density map (grey mesh) diagram of the function of AP-1 in the termination of STING signalling. One
of the pSTING tail is contoured at 3σ. g, HEK293T cells transfected with representative example of at least three (g–i) independent experiments is
Flag-tagged STING and HA-tagged wild-type AP-1σ or AP-1σ mutants σKR (K60A/ shown.
motif (in which ɸ denotes a hydrophilic residue) for IRF3 (refs. 17–19,27) by stronger IRF3 phosphorylation and more potent inhibition of HSV-1
(Fig. 3a). Of note, mutations of the two hydrophobic residues disrupted replication (Extended Data Figs. 4d and 6b). Given that the isoleucine
the binding of STING to AP-1 in activated cells, whereas mutation of the motif at position 365 (EXXXLI) is also required for the recruitment of
acidic residue alone had a negligible effect (Fig. 3b). A STING mutant IRF3 onto STING (ɸLXIS)17,27, mutagenesis of this position abolished the
devoid of the entire CTT also did not bind AP-1 (Fig. 3b). As expected activation of IRF3 (Extended Data Fig. 6c). Analysing natural variants
from the requirement of AP-1 for cargo loading, disruption of the LI of STING, we identified a missense mutation, p.LL363LF, located in the
motif abolished the incorporation of STING into CCVs and compro- EXXXLI motif that has been reported for human cancer39. Reconstitu-
mised subsequent degradation (Fig. 3c and Extended Data Fig. 6a). tion experiments revealed that the L364F substitution increased the
Moreover, neutralizing the hydrophobic leucine residue at position binding of STING to AP-1, accelerated STING degradation kinetics and
364 (EXXXLI) to alanine exaggerated type I IFN signalling, as shown strongly impaired STING signalling activity (Extended Data Fig. 6d–f).
Extended Data Fig. 5 | AP-1 depletion boosts STING-dependent immune N154S mutation (e,f), H72N (g,h) or V147M (i,j) were incubated with NC siRNA
activation in different cell types. a,b, HaCaT cells (a) or THP-1 cells or AP-1 siRNA for three days and then analysed by western blot. Vinculin was
(b) transfected with NC siRNA or AP-1 siRNA for 3 days were treated with 2.5 µM used as a loading control in f,h,j. e,g,i, Induction of IFNB1, IFIT1, IFIT2, IFIT3
diABZI for indicated time and analysed by western blot. GAPDH was used as a expression was assessed by RT–qPCR in HeLa cells transfected with NC siRNA
loading control. c,d, Human primary epithelial cells incubated with NC siRNA or AP-1 siRNA for 3 days and then stimulated with 1 µM diABZI. Mean ± s.d. of
or AP-1 siRNA for 3 days were treated with 2.5 µM diABZI and were analysed by three (c,e,g,i) technical replicates. P values based on two-tailed Student’s
RT–qPCR (c) and western blot (d). Vinculin was used as a loading control. t-tests (c,e,g,i).
e–j, Fibroblast cells derived from patients with SAVI characterized by STING1
Extended Data Fig. 6 | AP-1 binds to STING through a dileucine motif. analysed by western blot. e, HeLa cGAS/STING double KO cells transfected with
a, HeLa cGAS/STING double KO cells transfected with FLAG-tagged STINGWT, FLAG-tagged STINGWT or STINGL364F were treated or not with 2.5 µM diABZI for
STING1–317 or STINGLI(L364A/I365A) were treated or not with 2.5 µM diABZI for 14 h 14 h and analysed by western blot. GAPDH was used as a loading control. f, IFN-β
and analysed by western blot. Vinculin was used as a loading control. b, HeLa luciferase assay in HEK293T cells transfected with plasmids expressing indicated
cGAS/STING double KO cells transfected with FLAG-tagged STINGWT or STING constructions and stimulated with diABZI (2.5 µM). g, Glutathione
STINGL364A were treated or not with 2.5 µM diABZI for 14 h and analysed by Sepharose pull-down assays of LBD-STINGWT or LBD-STINGELI(E360A/L364A/I365A)
western blot. Vinculin was used as a loading control. c, HeLa cGAS/STING by GST-tagged AP-1 ΔμCTD core. One representative of three (a–c,e–f) or two
double KO cells transfected with FLAG-tagged STINGL363A, STINGL364A or (d,g) independent experiments is shown. Ratios of target proteins versus
STINGI365A were treated or not with 2.5 µM diABZI for 14 h and analysed by loading control normalized to the untreated sample of each condition (a–c,e).
western blot. GAPDH was used as a loading control. d, HEK293T cells Mean ± s.d. of three (f) technical replicates. P values based on two-tailed
transfected with FLAG-tagged STINGWT, STINGL374A or STINGL374F were treated Student’s t-tests (f).
with 2.5 µM diABZI for 2 h, immunoprecipitated with anti-FLAG antibody, and
Article
Extended Data Fig. 8 | Cryo-EM analysis of pSTING in complex with AP-1. pSTING complex, coloured according to the local resolution. g, 3D Fourier shell
a, Purification and SDS–PAGE analysis of AP-1 core in complex with pSTING and correlation of final 3D reconstruction of the AP-1 pSTING complex.
ARF1. b, Purification and SDS–PAGE analysis of AP-1 core in complex with h, Corrected Gold-standard Fourier shell correlation curves of the AP-1 pSTING
STING3S (S355D/S358D/S366D). c, Representative micrograph of AP-1 pSTING complex complex for the 3D electron microscopy reconstruction. i, Angular
in vitrified ice from 30,004 raw images. Scale bar, 30 nm. d, 2D class averages of distribution of the AP-1 pSTING particles included in the final
AP-1 pSTING complex particles. Box size, 27 nm. e, Flow chart of data reconstruction. One representative of at least two (a,b) independent
processing; see Methods for details. f, Final 3D reconstruction of the AP-1 experiments is shown.
Extended Data Fig. 9 | Density maps and structural models of the AP-1–pSTING complex. The density maps (grey mesh) of AP-1 and pSTING contoured at 3σ.
The protein structures fitted into the density map are shown by the stick models.
Article
Extended Data Fig. 10 | pS366 of pSTING binds to a basic patch of AP-1σ. FLAG-tagged STINGWT and HA-tagged AP-1γWT, γR15E, σWT, σI103S or σV88D,
a, Sequence alignment of AP-1σ in different species and human σ subunit respectively, were treated with 2.5 µM diABZI for 2 h, immunoprecipitated (IP)
isomers. b, Bio-layer interferometry binding studies of LBD-STING or with anti-FLAG antibody and then analysed by western blot. One representative
pLBD-STING with AP-1 ΔμCTD σKR. c, HEK293T cells transfected with of two (b,c) independent experiments is shown.
Extended Data Table 1 | Cryogenic-electron microscopy data summary table
μ μ μ