Science - USA (2020-08-21)

of a“pair of wings,”with an approximate
antiparallel four-helix bundle making up an
extensive buried dimer interface ( 19 , 20 ). The
solved cocrystal structures of cGAMP bound
to hSTING revealed that cGAMP binds in a
deep cleft at the dimer interface and causes
a substantial conformational change, which
leads to the formation of an antiparallel four-
strandb-sheet overhead cap element that
serves to completely envelop the CDN [fig. S7A,
( 18 , 21 )]. In the cGAMP-bound“closed”confor-
mation, the distance between the tips of the
symmetry-relateda2 helices is substantially re-
duced when compared with the unbound pro-
tein structure [fig. S7B; Protein Data Bank
(PDB) ID 4F9E ( 19 )]. By contrast, binding of
di-GMP CDN induces an increase in the angle
and distance between the tips of thea2heli-
ces, leading to the formation of an“open”con-
formation that lacks theb-sheet overhead cap
element [fig. S7C; PDB ID 4F9G ( 19 )]. Mech-
anistic and structural studies involving cGAMP
and DMXAA ( 18 , 21 ) suggested that ligands
that induce the closed conformation lead to
STING activation. Interestingly, diABZI-2 was
recently described as eliciting the phosphoryl-
ation of IRF3 and downstream target expres-
sion leading to antitumor immunity by binding
to STING and inducing an open conformation
akin to that induced by di-GMP [fig. S7D; PDB
ID 6DXL ( 22 )].ItisintriguingthatCDNspro-
duced directly by bacteria (i.e., di-GMP) can
activate STING by stabilizing an open con-
formation that is so substantially different to
that of the closed conformation, which is in-
duced by the endogenously produced second-
ary message of cytosolic double-stranded DNA
(i.e., cGAMP). The biological consequences of
differing CDN-induced conformation-dependent
STING scaffolding functions remain to be fully
characterized.
The 1.8-Å cocrystal structure of SR-717 bound
to hSTINGR232(residues 155 to 341) is shown
in Fig. 2A (supporting crystallography statis-
tics are shown in table S1), with simulated an-
neal omit maps of unbiased electron density
for bound ligands shown in fig. S8A. Binding
of two molecules of SR-717 at the base of the
STING dimer intersubunit cleft closely mimics
the binding mode of cGAMP. It induces the
same closed conformation (Fig. 2B and fig. S7A),
involving a near identical characteristic reduc-
tion in distance between the tips of thea 2
helices (Fig. 2C and fig. S7A), as well as the
formation of the four-strandb-sheet overhead
capelement(Fig.2,A,B,andD;andfig.S7A).
As occurs with both purine bases of bound
cGAMP ( 18 , 21 ), the pyridazine ring of each
molecule of SR-717 is“bracketed”by stacking
interactions with the side chains of Tyr^167 (Y167)
and Arg^238 (R238) from opposing monomers
(Fig. 2, D and F, and fig. S9A). The structure
of SR-717 facilitates a binding mode in which
Thr^263 (T263) side chain hydroxyls from both

Chinet al.,Science 369 , 993–999 (2020) 21 August 2020 3of7

A

SR-717

Y167a

R238a

cGAMP

SR-717a

SR-717b

Y167b

R238b

Y167a

SR-717a

SR-717b

R238 Y167b

b

R238a

T263b

T263a

Cap Element

37.8 Å

SR-717

di-GMP (4F9G)

37.8Å

54.7Å

SR-717

APO (4F9E)

37.8 Å

49.1 Å

SR-717

cGAMP (4KSY)

Cap Element (Closed)

B

CD

E F

Fig. 2. Crystal structure of SR-717 bound to hSTINGR232and comparison to existing hSTING

structures and intermolecular contacts.(A) The 1.8-Å structure of SR-717 bound to hSTINGR232(residues

155 to 341). Ribbon representation of the symmetrical hSTINGR232dimer and stick representations of

SR-717 are shown. (B) Superposition of the structure of SR-717 bound to hSTINGR232with the structure

of cGAMP bound to hSTING [PDB ID 4KSY ( 21 )], shown as yellow or blue ribbon representations, respectively,

highlighting near identical closed conformations and associated formation of characteristicb-sheet

overhead cap elements. Stick representations of bound SR-717 or cGAMP are shown. (C) Superposition

of the structure of SR-717 bound to hSTINGR232with the unbound (APO) structure of hSTING [PDB ID 4F9E

( 19 )], shown as yellow or purple ribbon representations, respectively. (D) Details of the intermolecular

contacts and hydrogen-bond (dashed red line) network associated with the binding of two molecules of

SR-717 to the cGAMP binding site of hSTINGR232. Residues from individual monomers of the hSTINGR232

dimer are shown in stick format in yellow and purple. The a and b superscripts indicate protein monomer or

individual ligand identity. (E) Superposition of the structure of SR-717 bound to hSTINGR232with the structure

of di-GMP bound to hSTING [PDB ID 4F9G ( 19 )], shown as yellow or orange ribbon representations,

respectively. Stick representations of SR-717 and di-GMP are shown. (F) Detailed view of the overlay and

conserved intermolecular contacts of bound SR-717 and cGAMP ligands from the overlaid hSTING structures

shown in (B). Side chains from the SR-717–bound hSTINGR232structure are shown in yellow or purple,

and those from the cGAMP structure are shown in blue. Stick representations of two molecules of SR-717 are

shown in gray, and cGAMP is shown in light blue.

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