Nature - USA (2020-10-15)

a

hi

036912

0.0

0.5

1.0

1.5

Time(h)

OD

600

induced, +NAM

uninduced, +NAM

uninduced,−NAM

induced,−NAM

SfSTING WT NAM supplementation

0 36912

0.0

0.5

1.0

1.5

Time(h)

OD

600

induced, +NAM

uninduced, +NAM

uninduced,−NAM

induced,−NAM

SfSTING D259A NAM supplementation

10 −3 10 −2 10 −1 100 101 102

0

2×10^5

4×10^5

6×10^5

8×10^5

[c-di-GMP] (μM)

ε-NAD

Cleavage

(RF

U)

WT

N163A K167A S262A

F165A R168A T263A

SfSTING mutant ε-NADturnover after 1 h

b

Residue mutated in SfSTING

(FsSTING equivalent)

Observed 3 ', 3 '-cGAMP

contact in FsSTING

N163 (N91)

F165 (F92)

K167 (F94)

R168 (P95)

D259 (D169)

S262 (N172)

T263 (T173)

R234 (R153)

Phosphodiester bond

Base stacking (outer face)

None/predicted only for SfSTING

None/predicted only for SfSTING

N2 of guanosine base

Phosphodiester bond

Potential clash with free 3 '-OH

of 2 '–5' linked CDNs

Base stacking (inner face)

Amino acids targeted for mutagenesis

f

1.52.0 2.53.0 3.5

0.0

0.5

1.0

Retention time (min)

Normalized absorbance 254 nm

WT Apo

WT + c-di-GMP

E84A + c-di-GMP

R234A + c-di-GMP

D259A + c-di-GMP

g

WT E84A R234A D259A

0

2×10^5

4×10^5

6×10^5

ε-NAD Cleavage (RFU

)

—+ —+ —+ —+

cd

c-di-AMPc-d

i-GMP

3',3'-cGAMP2',3'-cGAMPc-di-AMPc-di-GM

P

3',3'-cGAMP2',3'-cGAMP

SfSTING Mutant:R234A D259A

Free

CDN

[α^32 P] CDN:

Well

STING–CDN

Complex

STING:—

SfSTING D259A (TIR)

Free

c-di-GMP

Well

STING–CDN

Complex

100 nm[SfSTING D259A] (μM)

10 −3 10 −2 10 −1 100 101 102 103

0.0

0.4

0.8

1.2

Fraction

boun

d

c-di-GMP

WT fit

Kd= 28.78 μM (2 orders of magnitude shift)

e

Extended Data Fig. 6 | See next page for caption.