supplemental information structural analysis of the sting adaptor

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Immunity, Volume 36

Supplemental Information

Structural Analysis of the STING Adaptor Protein

Reveals a Hydrophobic Dimer Interface

and Mode of Cyclic di-GMP Binding Songying Ouyang, Xianqiang Song, Yaya Wang, Heng Ru, Neil Shaw, Yan Jiang, Fengfeng Niu, Yanping Zhu, Weicheng Qiu, Kislay Parvatiyar, Yang Li, Rongguang Zhang, Genhong Cheng, and Zhi-Jie Liu Inventory of Supplemental Material Supplemental Figures: Figure S1. STING CTD purified as a dimer. Related to Main Figure 1. Figure S2. The superposition of automatically traced model with Se-SAD phased 2Fo-Fc electron density map generated by RESOLVE. Related to Main Table 1. Figure S3. Model of the STING dimer showing the N-terminal transmembrane helices threading the membrane. Related to Main Figure 2. Figures S4. The structural view of STING CTD:c-di-GMP complex and its specificity analysis. Related to Main Figure 3. Figures S5. The screening of dimerization deficient mutants. Related to Main Figure 4. Figures S6. The binding assay of STING with small molecules by ITC and thermal shift and a model of STING S162Y:c-di-GMP complex structure. Related to Main Figure 6. Supplemental Tables: Table S1. Predicted STING transmembrane domains. Related to Main Figure 1. Table S2. Interactions between the two STING CTD molecules in homodimer (distances ≤ 5.0 Å). Related to Main Figure 2. Table S3. Interactions between c-di-GMP and the STING dimer including water (distances ≤ 4.0 Å). Related to Main Figure 3. Table S4. Thermal shift results. Related to Main Figure 6. Supplemental Experimental Procedures Supplemental References

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Figure S1. STING CTD purified as a dimer. (A) Strictly conserved residues are boxed in white on a red background and highly conserved residues are boxed in red on a white background. The 4 proposed transmembrane domains and the dimer interface are labeled in blue and red lines, respectively. Alignment was generated by ClustalW. The figure was generated by ESPript. (B) Phylogenetic tree was constructed according to multiple sequence alignment in A. (C) STINGΔ1-138 was loaded on a Superdex G75 size exclusion chromatography column (120 ml) and 1 ml fractions were collected. Fractions containing protein were run on SDS-PAGE. The boxed peak fractions (10-14) were pooled, concentrated, and set up for crystallization.

2nd peak

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Figure S2. The superposition of automatically traced model with Se-SAD phased 2Fo-Fc electron density map generated by RESOLVE (Terwilliger, 2003). The electron density map was calculated at 50.00 – 3.10 Å resolution, contoured at 1.0 σ.

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Figure S3. Model of the STING dimer showing the N-terminal transmembrane helices threading the membrane. (A) STING N-terminal aa 22-149 was modeled by the CPHmodels 3.0 homology modeling server (Nielsen et al., 2010) using the crystal structure of the bovine rhodopsin (PDB code 1F88-A) as a template. The color scheme is the same as in Figure 3A. The predicted last transmembrane region (aa 153-173), which is the hydrophobic dimer interface, is shown in magenta. Key residues, G158 (cyan), K150 (red), and other 3 mutation sites (V165, Y164, I165) (gray) are shown on one monomer of STING as sticks. Key residues for c-di-GMP binding, S162, Y167 and T263 are shown on another monomer of SITNG as slate sticks. C-di-GMP is shown as yellow sticks. Previously reported STING phosphorylation site S358 is shown in the C-terminal disordered region. The dashed line indicates missing residues. The N- and C-terminals of STING are labeled with residue numbers. (B) A top view of STING dimer model.

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Figure S4. The structural view of STING CTD:c-di-GMP complex and its specificity analysis. (A) Structural superposition of the liganded STING CTD dimer (pink) over unliganded (marine). C-di-GMP is shown as yellow sticks. Y240’s side chain of liganded STING CTD swings towards c-di-GMP as shown in stick. (B) Monomer A, monomer B and c-di-GMP are shown in orange, green and yellow, respectively. (C) Left panel: c-di-GMP bound with STING dimer; Right panel: c-di-AMP modeled into the STING dimer. C-di-AMP and c-di-GMP are shown as magenta and yellow sticks. STING prefers c-di-GMP over c-di-AMP due to the following facts: 1) Water 1 and 9 form hydrogen bonds with N2 and N21 of c-di-GMP separately. But similar hydrogen bonds are absent for c-di-AMP because there are no corresponding atoms (N2 and N21) in c-di-AMP (cyan rectangle); 2) The keto group of c-di-GMP is replaced by an amine group in c-di-AMP at C6 position that is opposite in charge. Water 2 and 3 were observed mediating hydrogen bonds between guanine and S241 (black rectangle). Hydrogen bonding atoms are connected by black dashed lines if the distance is less than 3.50 Å.

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Figure S5. The screening of dimerization deficient mutants. (A) Structure based mutants - V155R, G158L, W161A, Y164A, and I165R – were expressed in E. coli with an N-terminal 6His tag. The cyan color designates soluble mutants and the red ones designate insoluble mutants. The insoluble mutants V155R, W161A and Y164A were insoluble even when salvaged with an N-terminal GST tag. For each mutant 6 samples were loaded: the whole cells; the supernatant after sonication; the precipitate after sonication; flow-through of the Ni-column; 50 mM imidazole wash; after purification. The arrows designate position of the protein of interest. For GST fusion proteins 50 mM imidazole wash was omitted. (B) IFN-β reporter activity of 293T cells expressing STING WT and mutants. Error bars indicate SEM. (C) Co-immunoprecipitation of cell lysates from HEK 293T cells expressing STING WT and G158L mutant.

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Figure S6. The binding assay of STING with small molecules by ITC and thermal shift and a model of STING S162Y:c-di-GMP complex structure. (A) The affinity of STING for c-di-AMP measured by ITC. PBS was used as the negative control. (B) The binding study between STING CTD WT and mutants with c-di-GMP and c-di-AMP by thermal shift assay. The affinity of STING S162E (C) and S162Y (D) for c-di-GMP estimated by ITC. (E) A model of STING S162Y:c-di-GMP. C-di-GMP could not be docked in the trough of STING dimer interface due to steric hindrance. Residue Y162 was shown as spheres and dots. (F) pcDNA3 vector alone, pcDNA3-STING WT, and pcDNA3-STING S162Y were co-transfected into 293T cells together with the firefly luciferase reporter driven by the interferon β (IFN-β) promoter and renilla

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luciferase reporter. Cells were lysed at 48 hours after transfection. Luciferase activities were assayed using the Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer's instructions. The IFN-β promoter activities were normalized to renilla luciferase activities to control for transfection efficiency. The data presented in the figure were the averaged values with standard deviations from three independent transient transfections. Error bars indicate SEM. Table S1. Predicted STING transmembrane domains.

TM1 TM2 TM3 TM4 TM5 Source

21-39 45-67 88-108 112-136 153-173 (Ishikawa

and Barber, 2008)

20-36 47-66 87-103 114-133 154-171 (Sauer et al., 2011)

22-37 48-67 112-135 158-175 (Sun et al., 2009)

21-38 47-67 111-135 153-173 (Zhong et al., 2008)

21-38 87-109 121-141 153-172 (Xie et al., 2010)

21-43 84-106 116-138 155-178 (Jin et al., 2008)

21-41 47-67 116-136 153-173 UniProtKBa 21-37 46-65 96-114 119-136 155-172 HMMTOPb 17-37 47-67 112-136 153-173 SOSUIe 21-37 43-66 87-107 119-136 Phobiusc 18-40 45-67 SPLITd 21-36 45-66 95-108 116-139 TM Predf 22-34 47-63 96-107 118-136 DASg 20-40 47-67 110-130 TopPredi

45-64 86-107 118-134 psipredJ 21-37 47-67 117-136 PRED-TMRk 21-35 47-61 95-109 118-134 SVMtml

No TMs TMHMMh a http://www.uniprot.org/uniprot/Q86WV6 (accession code Q86WV6) b www.enzim.hu/hmmtop c http://phobius.sbc.su.se d http://split.pmfst.hr/split/4/ e http://bp.nuap.nagoya-u.ac.jp/sosui/ f www.ch.embnet.org/software/TMPRED_form.html g www.sbc.su.se/~miklos/DAS/maindas.html h www.cbs.dtu.dk/services/TMHMM i http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred j http://bioinf.cs.ucl.ac.uk/psipred/ k http://athina.biol.uoa.gr/PRED-TMR/input.html l http://ccb.imb.uq.edu.au/svmtm/svmtm_predictor.shtml

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Table S2. Interactions between the two STING CTD molecules in homodimer (distances ≤ 5.0 Å).

No. Molecule 1 Chain/residue

Molecule 2 Chain/residue

1. A/ASN152 B/ASN152 2. A/ASN152 B/ASN154 3. A/PHE153 B/HIS157 4. A/PHE153 B/TRP161 5. A/ASN154 B/HIS157 6. A/ASN154 B/ASN154 7. A/ASN154 B/GLY158 8. A/ASN154 B/ASN152 9. A/VAL155 B/HIS157 10. A/VAL155 B/GLY158 11. A/VAL155 B/TRP161 12. A/HIS157 B/VAL155 13. A/HIS157 B/ASN154 14. A/HIS157 B/PHE153 15. A/GLY158 B/GLY158 16. A/GLY158 B/LEU159 17. A/LEU159 B/GLY158 18. A/LEU159 B/SER162 19. A/TRP161 B/THR267 20. A/TRP161 B/VAL155 21. A/TRP161 B/MET271 22. A/TRP161 B/PHE153 23. A/TRP161 B/ALA277 24. A/TRP161 B/TYR274 25. A/SER162 B/THR267 26. A/SER162 B/LEU159 27. A/TYR164 B/TYR274 28. A/ILE165 B/THR267 29. A/ILE165 B/ALA270 30. A/ILE165 B/MET271 31. A/ILE165 B/TYR274 32. A/ARG169 B/TYR274 33. A/ARG169 B/ALA270 34. A/ARG169 B/GLN273 35. A/THR267 B/ILE165 36. A/THR267 B/TRP161 37. A/THR267 B/SER162 38. A/ALA270 B/ILE165 39. A/ALA270 B/ARG169 40. A/MET271 B/ILE165 41. A/MET271 B/TRP161 42. A/GLN273 B/ARG169 43. A/TYR274 B/ARG169

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44. A/TYR274 B/ILE165 45. A/TYR274 B/TYR164 46. A/TYR274 B/TRP161 47. A/TYR274 B/GLU304 48. A/TYR274 B/ALA302 49. A/TYR274 B/ILE298 50. A/TYR274 B/ASP301 51. A/GLN276 B/ASP301 52. A/GLN276 B/GLU304 53. A/ALA277 B/TRP161 54. A/ILE298 B/TYR274 55. A/ASP301 B/TYR274 56. A/ASP301 B/GLN276 57. A/ALA302 B/TYR274 58. A/GLU304 B/GLN276

The alternating gray and white background is intended to better distinguish different types of residues. Hydrophobic interactions are highlighted in red. Table S3. Interactions between c-di-GMP and the STING dimer including water (distances ≤ 4.0 Å).

No. c-di-GMP with chain A c-di-GMP with chain B c-di-GMP with water Interacting

atoms distance Interacting atoms distance Interacting

atoms distance

1. c-di-GMP/O2P A/THR 267/OG1 3.76 c-di-GMP/O2P

B/SER 162/O 3.80 c-di-GMP/P1 S/HOH 26/O 3.88

2. c-di-GMP/C1A A/TYR 163/CD1 3.86 c-di-GMP/O2P

B/GLY 166/CA 3.61 c-di-GMP/P11 S/HOH 34/O 3.71

3. c-di-GMP/C1A A/THR 263/OG1 3.45 c-di-GMP/C5'

B/SER 162/O 3.86 c-di-GMP/O1P S/HOH 41/O 3.05

4. c-di-GMP/C2A A/THR 263/OG1 3.58 c-di-GMP/C5'

B/SER 162/OG 3.07 c-di-GMP/O2P S/HOH 26/O 3.55

5. c-di-GMP/O2A A/THR 263/C 3.82 c-di-GMP/C4'

B/SER 162/OG 3.88 c-di-GMP/C3' S/HOH 34/O 3.90

6. c-di-GMP/O2A A/THR 263/CB 3.28 c-di-GMP/O4'

B/TYR 163/CA

3.87 c-di-GMP/O3' S/HOH 32/O 3.93

7. c-di-GMP/O2A A/THR 263/OG1 3.01 c-di-GMP/O4'

B/TYR 163/CD1 3.86 c-di-GMP/O3' S/HOH 52/O 3.14

8. c-di-GMP/O2A A/PRO 264/N 3.65 c-di-GMP/O4'

B/TYR 167/CB 3.91 c-di-GMP/C2' S/HOH 32/O 3.93

9. c-di-GMP/O2A A/PRO 264/CG 3.73 c-di-GMP/C2'

B/THR 263/OG1 3.54 c-di-GMP/O2' S/HOH 32/O 3.18

10. c-di-GMP/O2A A/PRO 264/CD 3.65 c-di-GMP/O2'

B/THR 263/C 3.79 c-di-GMP/O2' S/HOH 52/O 3.48

11. c-di-GMP/C4A A/SER 162/OG 3.88 c-di-GMP/O2'

B/THR 263/CB 3.41 c-di-GMP/N2 S/HOH 9/O 3.22

12. c-di-GMP/O4A A/TYR 163/CA 3.98 c-di-GMP/O2'

B/THR 263/OG1 3.02 c-di-GMP/O2A S/HOH 26/O 3.51

13. c-di-GMP/O4A A/TYR 163/CD1 3.72 c-di-GMP/O2'

B/PRO 264/N 3.53 c-di-GMP/C3A S/HOH 41/O 3.98

14. c-di-GMP/O4A A/TYR 167/CB 3.97 c-di-GMP/O2'

B/PRO 264/CG 3.51 c-di-GMP/O3A S/HOH 26/O 3.06

15. c-di-GMP/C5A A/SER 162/O 3.75 c-di-GMP/O2'

B/PRO 264/CD 3.46 c-di-GMP/C8 S/HOH 34/O 3.80

16. c-di-GMP/C5A A/SER 162/OG 3.04 c-di-GMP/C1'

B/TYR 163/CD1 3.84 c-di-GMP/N11 S/HOH 2/O 2.87

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17. c-di-GMP/N11 A/TYR 167/CD1 3.81 c-di-GMP/C1'

B/THR 263/OG1 3.59 c-di-GMP/O11 S/HOH 32/O 3.78

18. c-di-GMP/N11 A/TYR 167/CE1 3.27 c-di-GMP/N1

B/TYR 167/CD1 3.92 c-di-GMP/O11 S/HOH 34/O 2.63

19. c-di-GMP/N11 A/TYR 167/CZ 3.60 c-di-GMP/N1

B/TYR 167/CE1 3.41 c-di-GMP/C21 S/HOH 1/O 3.92

20. c-di-GMP/N11 A/TYR 167/OH 3.85 c-di-GMP/N1

B/TYR 167/CZ 3.77 c-di-GMP/C21 S/HOH 2/O 3.67

21. c-di-GMP/C21 A/TYR 167/CE1 3.67 c-di-GMP/C2

B/TYR 167/CE1 3.65 c-di-GMP/N21 S/HOH 1/O 2.92


B/TYR 167/CZ

3.63 c-di-GMP/N21

S/HOH 2/O 3.65

23. c-di-GMP/C21 A/TYR 167/CZ 3.62 c-di-GMP/C2

B/TYR 167/OH 3.98 c-di-GMP/N21 S/HOH 36/O 3.81

24. c-di-GMP/C21 A/TYR 167/OH 3.96 c-di-GMP/C2

B/THR 263/OG1 3.81 c-di-GMP/O21 S/HOH 32/O 3.76

25. c-di-GMP/C21 A/THR 263/OG1 3.77 c-di-GMP/N2

B/TYR 167/CZ 3.84 c-di-GMP/O21 S/HOH 52/O 3.66

26. c-di-GMP/N21 A/TYR 167/CZ 3.87 c-di-GMP/N2

B/TYR 167/OH 3.77 c-di-GMP/C41 S/HOH 62/O 4.00

27. c-di-GMP/N21 A/TYR 167/OH 3.82 c-di-GMP/N2

B/GLU 260/OE2 3.72 c-di-GMP/C51 S/HOH 62/O 3.62

28. c-di-GMP/N21 A/GLU 260/OE1 3.54 c-di-GMP/N2

B/THR 263/CG2 3.62 c-di-GMP/C61 S/HOH 2/O 3.72

29. c-di-GMP/N21 A/THR 263/CG2 3.58 c-di-GMP/N2

B/THR 263/OG1 3.72 c-di-GMP/C61 S/HOH 62/O 3.88

30. c-di-GMP/N21 A/THR 263/OG1 3.68 c-di-GMP/N3

B/TYR 167/CG 3.94 c-di-GMP/O61 S/HOH 2/O 3.73

31. c-di-GMP/O21 A/SER 162/O 3.94 c-di-GMP/N3

B/TYR 167/CD1 3.95 c-di-GMP/O61 S/HOH 3/O 2.93

32. c-di-GMP/O21 A/SER 162/OG 3.81 c-di-GMP/N3


33. c-di-GMP/O21 A/GLY 166/CA 3.72 c-di-GMP/N3

B/THR 263/OG1 3.02 c-di-GMP/N71 S/HOH 62/O 3.76

34. c-di-GMP/N31 A/THR 263/CB 3.93 c-di-GMP/C4

B/TYR 167/CG 3.73 c-di-GMP/C81 S/HOH 34/O 3.90

35. c-di-GMP/N31 A/THR 263/CG2 3.91 c-di-GMP/C4

B/TYR 167/CD1 3.67 c-di-GMP/C81 S/HOH 41/O 3.55

36. c-di-GMP/N31 A/THR 263/OG1 2.96 c-di-GMP/C4

B/THR 263/OG1 3.90 c-di-GMP/O5A S/HOH 34/O 3.73

37. c-di-GMP/C41 A/TYR 167/CG 3.81 c-di-GMP/C5


38. c-di-GMP/C41 A/TYR 167/CD1 3.71 c-di-GMP/C5

B/TYR 167/CE1 3.86

39. c-di-GMP/C41 A/THR 263/OG1 3.84 c-di-GMP/C6

B/TYR 167/CD1 3.63

40. c-di-GMP/C51 A/TYR 167/CG 3.96 c-di-GMP/C6

B/TYR 167/CE1 3.51

41. c-di-GMP/C51 A/TYR 167/CD1 3.40 c-di-GMP/N7

B/TYR 167/CD1 3.93


B/TYR 167/CB 3.99

43. c-di-GMP/C61 A/TYR 167/CD1 3.47 c-di-GMP/N9

B/TYR 167/CB 3.82

44. c-di-GMP/C61 A/TYR 167/CE1 3.31 c-di-GMP/N9

B/TYR 167/CG 3.94

45. c-di-GMP/O61 A/TYR 167/CD1 3.96 c-di-GMP/O21

B/THR 267/OG1 3.94

46. c-di-GMP/O61 A/TYR 167/CE1 3.71

47. c-di-GMP/O61 A/TYR 240/CD1 3.74

48. c-di-GMP/O61 A/TYR 240/CE1 3.89

49. c-di-GMP/N71 3.85

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A/TYR 167/CD1

50. c-di-GMP/N91 A/TYR 167/CB 3.96

Table S4. Thermal shift results.

proteins + c-di-GMP + c-di-AMP + PBS ΔTM1 ΔTM2 ΔTM1 ΔTM2 ΔTM1 ΔTM2

STING WT 3.85 3.69 0.12 0.09 0 0.01 STING K150R 5.87 ND ND ND 2.88 ND STING K150L 3.17 ND ND ND 0 ND STING K150A 5.59 ND ND ND 1.61 ND STING S358A 3.25 3.59 ND ND -0.40 -0.31 STING G344 3.82 3.84 ND ND 0.81 0.19 STING G158L Protein unstable STING I165R Protein unstable

ND: Not detected. ΔTM1 and ΔTM2 are repeated measurements of shifted temperature (°C) for key proteins and mutants. PBS: negative control.

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Supplemental Experimental Procedures

Protein Expression and Purification

Residues 139-379 and 149-379 of human STING were amplified from a

human transcription library (Stratagene, USA) and cloned into pMCSG7 vector

(Stols et al., 2002) for expression in E. coli BL21 (DE3). N-terminal

6His-tagged STING CTD was produced by growing the cells in LB media at

37 °C for 3 h until OD660nm reached 0.8. Induction was carried out at 16 °C for

24 h. Cells were harvested by centrifugation and re-suspended in a buffer

containing 50 mM PBS, pH 7.5, 10% glycerol and lysed by sonication. Soluble

STING CTD was purified by Ni affinity chromatography (Qiagen, USA) and

The protein was exchanged into a buffer containing 20 mM Tris-HCl, pH 7.5,

150 mM NaCl and and 1 mM DTT using a Superdex G75 (GE healthcare, USA)

gel filtration chromatography. After concentration using a 10 kD cut-off

centrifugal concentrator (Millipore, USA), the protein (15-20 mg/ml) was stored

in -80°C for biochemical assays or immediately screened for crystallization.

TBK1 used in the GST pull-down assay was purchased from OriGene

Technologies (TP305238).

Crystallization and Data Collection

The initial crystallization conditions were screened using commercially

available sparse-matrix screening kits from Hampton Research (Crystal

Screens 1 and 2, Index and PEG/ION Screens) and Emerald Biosystems

(Wizard I - IV) by robot. Hits were optimized by hand. 2 µl hanging drops

containing 1 µl protein mixed with 1 µl mother liquor were equilibrated over 300

µl reservoir solution and incubated at 16 °C. Unliganded STING CTD proteins

crystallized in 14.4% (w/v) PEG 8000, 0.08 M Cacodylate, pH 6.5, 0.16 M

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calcium acetate and 20% (v/v) glycerol in space group C2221, diffracted X-rays

to 2.45 Å at beamline BL17U1 of Shanghai Synchrotron Radiation Facility

(SSRF), China. The STING CTD and c-di-GMP complex was formed by mixing

eqi-molar amounts of STING CTD with c-di-GMP (Biolog, USA). The complex

and Se-Met STING crystallized in the same crystallization condition as that of

unliganded STING CTD.

Structure Determination and Analysis

Se-Met, native and STING CTD with c-di-GMP binary complex diffraction data

were collected at wavelength of 0.9793 Å. Data were indexed and scaled using

HKL2000 (Otwinowski Z., 1997). The initial phases were determined by

Se-SAD (single-wavelength anomalous dispersion) method (Hendrickson,

1991). PHENIX AutoBuild was used to rebuild the model with the initial phase

(Terwilliger et al., 2008). Other structures were solved by molecular

replacement method with program Phaser (McCoy et al., 2007). The models

were manually improved in Coot (Emsley and Cowtan, 2004). Refinement was

carried out using REFMAC (Murshudov et al., 1997) and PHENIX (Adams et

al., 2010) alternately. The quality of the final model was validated with

MolProbity (Chen et al., 2010). Structures were analyzed using PDBePISA

(Protein Interfaces, Surfaces and Assemblies) (Krissinel and Henrick, 2007),

Dali (Holm and Rosenstrom, 2010), ProFunc (Laskowski et al., 2005).

Isothermal Titration Calorimetry (ITC)

ITC measurements were performed on an iTC200 calorimeter (Microcal Inc.,

Northampton, MA). All experiments were carried out at 20 °C in 20 mM

Tris-HCl, pH 7.5, 150 mM NaCl. The reactant (50 µM STING CTD) was placed

in the 300 µl sample chamber and c-di-GMP (500 µM) was added using the

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syringe with 20 successive additions of 2 µl for 4 s (with an initial injection of

0.5 µl). The interval between each injection lasted 120 s. The peaks generated

were corrected for c-di-GMP heat of dilution and integrated using the ORIGIN

software (Microcal Inc) by plotting the values in microcalories against the ratio

of total moles of injectant, c-di-GMP, to reactant STING CTD, within the cell.

Data were fit using a 2:1 STING CTD: c-di-GMP binding model. All ITC

experiments were repeated independently at least two times and had less than

5% error in the KD values and the thermodynamic parameters between

experiments. Values from one experiment are presented.

GST Pull-down

GST and GST fusion protein (STING CTD mutants) were expressed in E. coli

and conjugated to glutathione-agarose beads. The amounts of GST and GST

fusion proteins on the beads were normalized by running SDS-PAGE and

staining with Coomassie brilliant blue R-250. GST and GST-fusion proteins

were incubated with 1.65 μg of recombinant TBK1 (OriGene Technologies,

TP305238) with or without c-di-GMP and c-di-AMP in a final volume of 1.0 ml

of PBS (137.0 mM NaCl, 2.7 mM KCl, 50.0 mM Na2HPO4,10.0 mM KH2PO4,

pH 7.4) with 2 mM DTT, 1 mM EDTA, and 1% Nonidet P-40 at 4 °C for 2 h. The

beads were washed five times (1 ml each) with the incubation buffer with 1.0%

NP-40 and subjected to SDS-PAGE analysis followed by immunoblotting with

anti-Flag polyclonal antibody (OriGene Technologies, TA150014).

Thermal Shift

Thermal shift assays were conducted using 0.04 mg/ml of STING CTD or its

mutations with or without 0.2 mM of c-di-GMP/c-di-AMP/GTP/dGTP in PBS

(137.0 mM NaCl, 2.7 mM KCl, 50.0 mM Na2HPO4,10.0 mM KH2PO4, pH 7.4),

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supplemented with or without 0.2 mM MgCl2, and a 1000 dilution of SYPRO

Orange dye (Invitrogen) as described previously by (Lavinder et al., 2009). The

fluorescence signals as a function of temperature were recorded using a Real

T-time PCR machine (Bio-Rad CFX96) in the FRET mode, in which the

fluorescence intensity was measured with Ex/Em: 450-490/560-580 nm. The

temperature gradient was set in the range of 20-95 °C with a ramp of 0.5 °C

over the course of 15 s. Control assays were carried out with buffer in the

presence or absence of c-di-GMP. Data were analysed with the differential

scanning fluorimetry analysis tool (Excel based), and the Boltzmann model

was used for plotting melting curves of STING to obtain the midpoint of the

thermal unfolding value (Tm) for STING using the curvefitting software XLfit 5

(www.idbs.com, ID Business Solutions Ltd).

Analytical Ultracentrifugation

Sedimentation velocity experiments were performed on a Beckman XL-I

analytical ultracentrifuge at 20 °C. Protein samples were diluted with PBS to

400 μl at a concentration of about 0.9 mg/ml. Samples were loaded into a

conventional double sector quartz cell and mounted in a Beckman 4-hole

An-60 Ti rotor. Data were collected at 60,000 r.p.m. at a wavelength of 280 nm.

Interference sedimentation coefficient distributions, c(M), were calculated from

the sedimentation velocity data by using SEDFIT

(www.analyticalultracentrifugation.com).

Cells and Reagents

HEK 293T cells were maintained in DMEM (Gibco) supplemented with 10%

FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. For retroviral infection,

HEK 293T cells were transfected with helper construct plus either pBABE

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alone or the indicated pBABE-STING constructs. Tmem173−/− MEFs, (a gift

from G.N. Barber (University of Miami, Florida, USA)), were then infected with

the filtered 293T cell supernatants and stably expressing cell lines were

selected in the presence of 2.5 µg/ml puromycin. B-DNA (Poly(dA:dT);

Invivogen) was transfected into cells at 1 µg/ml using Lipofectamine 2000

(Invitrogen). Antibodies against HA was purchased from Santa Cruz. All other

reagents were purchased from Sigma unless otherwise stated.

Immunoblotting and Immunoprecipitation

HEK 293T cells were transfected with indicated plasmids using Lipofectamine

2000. Forty-eight hours post transfection, cells were homogenized for 30 min

at 4 °C in a modified radioimmune precipitation buffer containing 0.1% (vol/vol)

Triton X-100 and no SDS. Protease inhibitor cocktail (Roche) was included in

all lysates. Cell lysates were then incubated with anti-Flag M2 affinity gel or

anti-HA affinity gel (Sigma) for 2 h at 4 °C and the immunoprecipitated

complexes were separated by SDS-PAGE and blotted with indicated

antibodies.

IFN-β Luciferase Reporter Assay

HEK 293T cells were transfected with IFN-β firefly luciferase and renilla

luciferase reporter plasmids together with STING WT or STING mutants.

Forty-eight hours post transfection, firefly and renilla luciferase activities were

determined using a Dual Luciferase Assay System (Promega) and a SIRIUS

Luminometer (Berthold Detection Systems) according to the manufacturer’s

protocol.

Real-time Quantitative PCR

RNA was isolated using TRIzol (Invitrogen), and cDNA was synthesized using

22

iScript (Bio-Rad). Quantitative real-time PCR (Q-PCR) analysis was performed

using the LightCycler 480 System (Roche). All gene expression data

presented were normalized to GAPDH levels for each sample, and fold

expression was determined as relative to unstimulated samples.

Statistical Analysis

The significance of differences between groups showing similar variance was

evaluated by the Student’s t-test.

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supplemental information structural analysis of the sting adaptor

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