Nature - USA (2020-10-15)

d

a

c

h

02468

0

500

1000

1500

2000

2500

Retention time (min)

Absorbance 254 nm (mAU

)

cADPr

ADPr

NAD+

NAM

01234567

0

100

200

300

400

500

Retention time (min)

Absorbance 254 nm (mAU

)

cADPr

ADPr

NAD+

NAM

01234567

Retentiontime(min)

ADPr

NAD+

NAM

Apo

c-di-AMP

c-di-GMP

3',3'-cGAMP

2',3'-cGAMP

b

100 Å

02468

0

500

Retention time (min)

Absorbance 254 nm (mAU)

c-di-GMP

Apo

ADPr

NAD+

NAM c-di-GMP

i

10 -3 10 -2 10 -1 100 101 102 103

0

100

200

300

400

500

[c-di-GMP] (μM)

ε-NAD cleaved (μM)

j k

0

2×10^4

4×10^4

6×10^4

8×10^4

Time (min)

ε-NAD Cleavage (RFU

) Glutathione resin + 0.5 μM protein

Glutathione resin + 2.0 μM protein

Ni2+ resin + 0.5 μM protein

Ni2+ resin + 2.0 μM protein

010203 0

SfSTING GST-TIR (Δ CDN-binding domain)

SfSTING SUMO-TIR (Δ CDN-binding domain)

0

2×10^4

4×10^4

6×10^4

8×10^4

Time (min)

ε-NAD Cleavage (RFU

)

100 μM20 μM

4 μM

800 nM

160 nM32 nM

6.4 nM

1.28 nM

Apo

010203040

SfSTING GST-TIR (Δ CDN-binding domain)

Protein Titration

Apo

c-di-AMPc-di-GMP3',3'-cGAMP2',3'-cGAMP

0

25

50

75

100

%A

reaA

DPr/

To talADPr & NA

+D

2.02.2 2.42.6 2.83.0

0

250

500

750

1000

Retention time(min)

Absorbance

254

nm

(mAU)

2',3'-c-di-GMP at 1h

2',3'-c-di-GMP at 2h

2',3'-c-di-GMP at 3h

3',3'-c-di-GMP at 5min

2.02.2 2.42.6 2.83.0

0

400

800

1200

Retentiontime(min)

Absorbance

254

nm

(mAU)

Apo

10 μM

3',3'-c-UMP–AMP

284 μM

3',3'-c-UMP–AMP

SfSTING NAD+ turnover after 180 min

fg

e

010203

04

040

0

2×10^5

4×10^5

6×10^5

8×10^5

Time(min)

ε-NADC

leavag

e(RFU)

c-di-GMP titration

Apo

100nM

10 nM

500nM

50 nM

10 μM 100 μM

20 nM

N N

N

NH 2

N

O

OHOH

P O

O-

O

P O

O-

O

O

OHOH

N+

NH 2

O

O

TIR

1000

Extended Data Fig. 5 | See next page for caption.