Binding-Pocket and Lid-Region Substitutions Render Human

Report
Binding-Pocket and Lid-Region Substitutions Render
Human STING Sensitive to the Species-Specific Drug
DMXAA
Graphical Abstract
Authors
Pu Gao, Thomas Zillinger, ..., Winfried
Barchet, Dinshaw J. Patel
Correspondence
[email protected] (W.B.),
[email protected] (D.J.P.)
In Brief
The anticancer drug DMXAA specifically
activates the STING pathway in a species-dependent manner. Gao et al.
combine calorimetric, structural, and
cellular studies to investigate the mechanism underlying DMXAA species selectivity. They find a critical role for a lid residue at position 230 and unveil the
structural basis for the mouse specificity
of DMXAA. This work also suggests
ways to render human STING responsive
to DMXAA.
Highlights
Accession Numbers
Residues critical for species-selective STING sensitivity to
DMXAA are uncovered
4QXO
4QXP
4QXQ
4QXR
S162A/Q266I substitutions endow hSTING with the same
DMXAA sensitivity as mSTING
The triple mutant S162A/G230I/Q266I renders hSTING highly
sensitive to DMXAA
Gao et al., 2014, Cell Reports 8, 1668–1676
September 25, 2014 ª2014 The Authors
http://dx.doi.org/10.1016/j.celrep.2014.08.010
Cell Reports
Report
Binding-Pocket and Lid-Region Substitutions
Render Human STING Sensitive
to the Species-Specific Drug DMXAA
Pu Gao,1,5 Thomas Zillinger,3,5 Weiyi Wang,2 Manuel Ascano,4 Peihong Dai,2 Gunther Hartmann,3 Thomas Tuschl,4
Liang Deng,2 Winfried Barchet,3,* and Dinshaw J. Patel1,*
1Structural
Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
3Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn 53127, Germany
4Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065, USA
5Co-first author
*Correspondence: [email protected] (W.B.), [email protected] (D.J.P.)
http://dx.doi.org/10.1016/j.celrep.2014.08.010
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
2Dermatology
SUMMARY
The drug DMXAA (5,6-dimethylxanthenone-4-acetic
acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to
activate mouse, but not human, STING, an adaptor
protein in the cyclic dinucleotide cGAMP-mediated
signaling pathway, inducing type I interferon expression. To facilitate the development of compounds
that target human STING, we combined structural,
biophysical, and cellular assays to study mouse
and human chimeric proteins and their interaction
with DMXAA. We identified a single substitution
(G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive ‘‘open’’
to an active ‘‘closed’’ state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A
binding-pocket point substitution, rendering hSTING
highly sensitive to DMXAA. These findings should
facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and
their exploitation for vaccine-adjuvant and anticancer drug development.
INTRODUCTION
The endoplasmic reticulum transmembrane protein STING
(stimulator of interferon genes) (Ishikawa and Barber, 2008; Ishikawa et al., 2009; Jin et al., 2008; Sun et al., 2009; Zhong et al.,
2008) is a central player in the innate immune response to cytosolic double-stranded DNA (Burdette and Vance, 2013). STING,
which responds to various forms of pathogen-derived DNA, as
well as to self-DNA, functions as an adaptor protein that recruits
and activates TANK binding kinase (TBK1) and IkB kinase (IKK),
which, following their phosphorylation, activate nuclear tran-
scription factors interferon regulatory factor 3 (IRF3) and nuclear
factor kappa B (NF-kB), respectively. STING was shown to be a
direct sensor of bacterial cyclic dinucleotides (CDNs) (Burdette
et al., 2011), although it was subsequently demonstrated that
the host-encoded cytosolic DNA-sensor cyclic GMP-AMP synthase (cGAS) (Sun et al., 2013) produces the second messenger
cyclic GMP-AMP (cGAMP) (Wu et al., 2013), which then binds
and activates STING. Independent studies by several groups
demonstrated that a noncanonical cGAMP linkage isomer,
c[G(20 ,50 )pA(30 ,50 )p], is produced by cGAS upon DNA binding
(Ablasser et al., 2013; Diner et al., 2013; Gao et al., 2013a; Zhang
et al., 2013). Follow-up structure-function studies showed that
human and mouse STING (hSTING and mSTING, respectively)
undergoes an ‘‘open’’ to ‘‘closed’’ conformational transition
upon binding c[G(20 ,50 )pA(30 ,50 )p] (Gao et al., 2013b; Zhang
et al., 2013). Our studies have primarily focused on the R71/
G230/R232/R293 variant of hSTING (hSTINGR232).
The xanthenone derivative compound DMXAA (Vadimezan,
5,6-dimethylxanthenone-4-acetic acid; Figure 1A) was initially
identified as a small molecule that exhibits immune modulatory
activities through the induction of cytokines and disrupts tumor
vascularization in multiple mouse models (Baguley and Ching,
2002). DMXAA in combination with paclitaxel and carboplatin
was evaluated in a phase II clinical trial against non-small-cell
lung cancer, but ultimately failed in human phase III trials (Lara
et al., 2011). Recently, it was demonstrated that DMXAA-induced
interferon-b (IFN-b) production by murine macrophages is dependent on STING, suggesting that mSTING is the protein target of
DMXAA (Prantner et al., 2012). Despite the high sequence identity
between mSTING and hSTING (68% amino acid identity and 81%
similarity) (Diner et al., 2013), DMXAA activates mSTING but has
no effect on hSTING (Conlon et al., 2013; Kim et al., 2013), which
hampers DMXAA’s therapeutic potential in humans.
Our earlier structure-function studies revealed that mSTING
binds to DMXAA using the same pocket as the natural c
[G(20 ,50 )pA(30 ,50 )p] and induces a similar ‘‘open’’ to ‘‘closed’’
conformational transition (Gao et al., 2013b). Given that identical
residues line the DMXAA binding pocket of both mSTING and
hSTING, it is unclear why DMXAA only activates mSTING.
Following our initial observation that a point substitution
1668 Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors
A
B
Figure 1. Replacement of Nonconserved
Residues of hSTING with Its Murine Counterparts Enables Recognition of DMXAA as
well as the Crystal Structure of DMXAA
Bound to hSTINGgroup2
C
O
3
2
1
OH
4
O
O
hSTINGgroup1234
5
8
hSTINGgroup2
CH3
6
7
CH3
untreated
DMXAA
D
IFN -Gluc, RLU
0.6
0.4
0.2
4
3
12
up
up
ro
ro
Gg
Gg
TI
N
N
hS
hS
TI
N
TI
hS
12
4
4
13
ro
Gg
N
Gg
ro
up
23
up
12
up
ro
Gg
TI
N
TI
hS
N
TI
hS
hS
34
4
3
up
Gg
ro
up
2
Gg
N
hS
TI
TI
hS
ro
up
1
N
Gg
ro
up
G
ro
N
Gg
sm
TI
hS
TI
N
hS
la
lP
C
on
tro
m
ST
IN
id
G
0.0
G Y240
E
hSTINGgroup2
+
DMXAA
F
R238
Y240
Y240 R238
I235
G230I R238
I171
I235
L170
L170
Q266
T263
L170
Y240
Q266
I171
T263
I165
T267
R232
I235
(A) Chemical formula of DMXAA.
(B and C) ITC binding curves for complex formation between DMXAA bound to hSTINGgroup1234
(aa 140–379) (B) and hSTINGgroup2 (C).
(D) 293T cells were transfected with IFN-b reporter
constructs and STING variants as indicated. At
12 hr after transfection, cells were stimulated with
0.18 mM DMXAA (50 mg/ml). Luciferase activity
was determined after another 12 hr. Dotted lines
separate (from left to right) WT controls, single
group mutants, hSTINGgroup1234, and triple-group
mutants. Shown are raw values of Gaussian
luciferase activity normalized to constitutive Firefly
luciferase values. Values depicted are the means
of triplicates + SEM and are representative of three
independent experiments.
(E) The 1.88 A˚ crystal structure of DMXAA bound to
hSTINGgroup2 (aa 155–341). The symmetrical
hSTINGgroup2 dimer is shown in a ribbon representation, with individual monomers colored in
yellow and magenta. The DMXAA (in a spacefilling representation) is bound in the central cavity
at the interface between the two monomers.
(F) Intermolecular contacts in the complex. The
bound DMXAA is shown in biscuit color, with individual STING subunits in the symmetrical dimer
shown in yellow and magenta.
(G) Two expanded views of the hydrophobic interactions of the G230I substitution (in green) in the
complex (blue box region in E). Other residues
lining the hydrophobic pocket are shown in yellow.
See also Figures S1 and S2.
R238
G230I
RESULTS
I235
S162 S162
I165
T267
(S162A) of hSTING placed within the CDNs/DMXAA binding site
rendered it partially sensitive to DMXAA (Gao et al., 2013b), we
reasoned that either smaller substituents or slightly modified
DMXAA variants could be promising candidates for the activation of hSTING and have potential for development as anticancer
drugs or vaccine adjuvants.
Here, we describe our detailed investigation of the mechanism
of DMXAA species selectivity through a combination of structural, biophysical, and cellular techniques. Our studies establish
that Q266I binding-pocket and G230I lid substitutions, together
with the previously identified binding-pocket S162A substitution,
rendered hSTING highly sensitive to DMXAA. These findings
provide a critical guide for future rational drug design of DMXAA
variants with potential IFN-b-stimulating activity in humans,
which are needed for the development of anticancer therapies
and vaccine adjuvants.
The Lid Region of the Ligand
Binding Pocket Is Important for
DMXAA Recognition
Within STING, DMXAA (Figure 1A) and c
[G(20 ,50 )pA(30 ,50 )p] share the same ligand
binding pocket (Gao et al., 2013b), which in human and mouse
proteins is composed of identical amino acids. Despite the fact
that the hSTING and mSTING C-terminal domains (CTD, aa
140–379) exhibit 76% amino acid identity (Figure S1), DMXAA
only binds and activates mSTING, and has no effect on hSTING
(Conlon et al., 2013; Kim et al., 2013). Therefore, the nonconserved residues between the two species that are located
outside the DMXAA binding pocket must play a role in distinct
DMXAA recognition. Guided by the available structural information on STING-ligand complexes (Gao et al., 2013b), we subdivided the nonconserved residues located in the STING CTD
into four groups (groups 1–4). We then substituted hSTING
residues with their mSTING counterparts for each of the four
groups (Figure S1). These residues are located either along
the dimer interface or within the regions that undergo large
conformational changes during the ‘‘open’’ to ‘‘closed’’ transition
L170
R232
Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors 1669
associated with complex formation. We also generated a
construct containing the combined substitution in all four groups
(hSTINGgroup1234).
We performed isothermal titration calorimetry (ITC) experiments to measure the DMXAA binding affinity of hSTING
CTD (aa 140–379) containing various group substitutions.
hSTINGgroup1234 showed a comparable exothermic binding
curve and binding affinity (KD: 0.69 mM) (Figure 1B) to mSTING
(KD: 0.49 mM) (Gao et al., 2013b). Similar to what was found for
wild-type (WT) hSTING protein, no detectable binding to DMXAA
was observed for the isolated group1, group3, or group4 substitutions of hSTING (Figure S2A). Only group2 substitutions of
hSTING exhibited detectable endothermic binding with DMXAA
(KD: 3.12 mM; Figure 1C).
To validate the binding results, we used an IFN-b luciferase reporter assay to further test the responsiveness of hSTING group
substitutions to DMXAA stimulation in human 293T cells, which
lack endogenous STING expression. For this cellular assay, we
used full-length hSTING (WT and substitutions) and mSTING
(WT) constructs, which were expressed at moderate levels to
allow ligand-dependent activation of the IFN-b promoter. We
confirmed that mSTING-transfected 293T cells responded to
DMXAA, whereas hSTING-transfected cells did not (Figure 1D,
left panel). Consistent with the ITC results, among the individual group substitutions, only the hSTINGgroup2 substitutions
showed responsiveness to DMXAA (Figure 1D, middle panel).
Inversely, removing the group2 substitutions from the combined
group1234 substitutions (hSTINGgroup134) strongly diminished
DMXAA activation, whereas loss of any of the other groups
was tolerated (Figure 1D, right panel). These results indicate
that group2 residues from mSTING, which are located within
the lid region of the binding pocket, play an important role in
DMXAA recognition.
Crystal Structure of DMXAA Bound to hSTINGgroup2
We proceeded to solve the crystal structure of DMXAA bound to
hSTINGgroup2 (aa 155–341) at 1.88 A˚ resolution (for X-ray statistics, see Table S1) with the complex containing two molecules of
DMXAA per hSTINGgroup2 dimer (Figure 1E). The results were
similar to what we had previously observed for the complex of
mSTING and DMXAA (Gao et al., 2013b). The four-stranded,
antiparallel, b-pleated sheet formed a lid covering the binding
pocket, indicative of the formation of a ‘‘closed’’ conformation
of STING upon complex formation. The aromatic rings of the
two DMXAA moieties were aligned in parallel, with complex formation mediated by both intermolecular van der Waals contacts
and hydrogen-bond interactions (Figure 1F). We observed excellent superposition of hSTINGgroup2 and mSTING in their complexes with DMXAA, as shown in Figure S2B (root-mean-square
deviation [rmsd]: 0.95 A˚).
To elucidate the molecular basis underlying DMXAA species
selectivity, we compared the structure of the hSTINGgroup2DMXAA complex with that of the mSTING-DMXAA complex
(Gao et al., 2013b). We found that in the hSTINGgroup2-DMXAA
structure, the side chain of the substituted residue I230 (G230
in WT protein) is located in a hydrophobic pocket composed of
residues from both the four-stranded, antiparallel b-sheet region
(R232, I235, R238, and Y240) and the adjacent long a-helix (L170
and I171) (Figure 1G). The amino acids that form the hydrophobic
pocket are identical between human (Figure 1G) and mouse
(Figure S2C) proteins. This isoleucine-mediated hydrophobic
interaction may help stabilize the b sheet and other parts of the
protein, facilitating DMXAA-mediated formation of the ‘‘closed’’
conformation by mSTING or hSTINGgroup2, thereby explaining
the absence of complex formation by WT hSTING with a glycine
at this position.
G230 of hSTING and I229 of mSTING Are Critical
Contributors to Differential DMXAA Recognition
To support our conclusions based on our structural findings
described above, we generated the G230I single substitution in
hSTING and tested its IFN-b induction activity using the luciferase assay. Indeed, hSTINGG230I alone was sufficient to mimic
the effects observed for hSTINGgroup2, resulting in an induction
of IFN-b almost identical to that found for hSTINGgroup2 (Figure 2A). Using the same method, we also generated and tested
reverse substitutions on mSTING (I229G or I229A). As expected,
mSTINGI229G and mSTINGI229A showed a significant decrease in
DMXAA-mediated IFN-b induction (Figure 2B).
We also solved the crystal structure of DMXAA bound to
hSTINGG230I (aa 155–341) at 2.51 A˚ resolution (X-ray statistics
in Table S1), with hSTINGG230I in the complex forming a ‘‘closed’’
conformation (Figure 2C). The detailed intermolecular contacts
in the complex (Figure S3A) are similar to those observed for
the hSTINGgroup2-DMXAA structure (Figure 1F). We observed
excellent superposition of hSTINGG230I and hSTINGgroup2 in their
complexes with DMXAA, as shown in Figure S3B (rmsd: 0.61 A˚).
The I230 residue, which is positioned within a hydrophobic
pocket (Figure 2D), forms the same intramolecular contacts as
observed in the structures of the hSTINGgroup2-DMXAA (Figure 1G) and mSTING-DMXAA (Figure S2C) complexes. Taken
together, our structural and functional data strongly demonstrate
that the substitution of Gly with Ile at position 230 results in the
gain of function of hSTING for DMXAA recognition.
hSTINGQ266I Is Activated by DMXAA
Guided by the structures of complexes of hSTING substitutions
with DMXAA, we next tested additional substitutions within the
ligand binding pocket to identify more constraints that would
help in the design of future modifications on DMXAA. We generated five substitutions (G166S, I235L, Q266I, Q266L, and
Q266V) in hSTING (Figure S1) to either enhance the hydrophobic
interaction or introduce additional hydrogen bonds with DMXAA.
The initial IFN-b induction results showed that only the Q266I
substitution in hSTING conferred DMXAA sensitivity at a level
similar to that previously observed for the S162A substitution
(Gao et al., 2013b; Figure 3A). Q266L resulted in a less pronounced gain of DMXAA-mediated IFN-b induction, whereas
G166S, I235L, and Q266V showed no effects (Figure 3A). We
next tested whether the S162A/Q266I double substitution
would augment DMXAA recognition, and indeed observed an
enhanced DMXAA-induced IFN-b induction similar to that
found for mSTING (Figure 3B). These results were confirmed
by ITC studies, which showed that hSTINGS162A/Q266I binds to
DMXAA with higher affinity (KD: 1.99 mM; Figure 4C) than either
hSTINGS162A (Figure S3C) or hSTINGQ266I (Figure S3D).
1670 Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors
B
0.6
IFN -Gluc, RLU
1.5
0.4
0.5
23
GG
0.0
3
IN
Gg
1.0
10
30
100
DMXAA (µM)
hS
T
IN
hS
T
C
Figure 2. G230 in hSTING and I229 in
mSTING Are Essential for DMXAA Species
Selectivity
mSTING
mSTINGI229G
mSTINGI229A
0I
2
up
ro
G
IN
IN
m
ST
0.0
DMXAA
(µM)
266
G 89
0
0.2
hS
T
IFN -Gluc, RLU
A
hSTINGG230I
+
DMXAA
D
Y240
R238
I171
G230I
I235
(A) 293T cells were transfected with IFN-b reporter
constructs and STING variants as indicated. At
12 hr after transfection, cells were stimulated with
ascending concentrations of DMXAA. Luciferase
activity was determined after another 12 hr.
Shown are the means of triplicates + SEM,
representative of three independent experiments.
(B) 293T cells were transfected with mSTING
variants and reporter constructs. Stimulation and
luciferase assay were performed as described in
(A). Dose responses are representative of two independent experiments.
(C) The 2.51 A˚ crystal structure of DMXAA bound
to hSTINGG230I (aa 155–341). The representations
and color codes are the same as in Figure 1E.
(D) Detailed hydrophobic interactions in the
complex of DMXAA bound to hSTINGG230I, with
the same representations and color codes as in
Figure 1G.
See also Figure S3.
L170
gands (Figure 3F). The crystal structures
of hSTINGS162A/Q266I and hSTINGG230I in
their bound complexes with DMXAA superimpose with an rmsd of 0.70 A˚ (Figure S4C). The details of the intermolecular contacts in the
complex are shown in Figure S4D, with the same intermolecular
hydrogen-bonding interaction network as observed in the
hSTINGgroup2-DMXAA (Figure 1F) and hSTINGG230I-DMXAA (Figure S3A) complexes. The substituted I266 side chain forms a hydrophobic patch together with the side chains of I165, L170, and
I235, which fully covers the aromatic methyl groups (positions 5
and 6) and the nonsubstituted aromatic edges (positions 7 and 8)
of DMXAA (Figure 3G). The substituted A162 side chain is juxtaposed with the aromatic edges lining the other side (positions 1
and 2) of DMXAA, forming additional hydrophobic interactions
(Figure 3G). S162A and Q266I substitutions increase the binding
affinity between hSTING and DMXAA and apparently help
hSTING to overcome the energy barrier when transitioning
from an ‘‘open’’ to a ‘‘closed’’ conformation.
R232
Besides the prevalent allelic hSTING variant (R71/G230/R232/
R293, hSTINGR232), four major nonsynonymous variants are
found with high frequencies in the human population: R71H/
G230A/R293Q (hSTINGHAQ), 20.4%; R232H (hSTINGH232),
13.7%; G230A/R293Q (hSTINGAQ), 5.2%; and R293Q
(hSTINGQ293), 1.5% (Yi et al., 2013). To determine whether the
S162A and Q266I substitutions were effective in all natural
hSTING variants, we generated the respective single and double
substitutions for all major hSTING alleles (listed in Figure 3D) and
tested them for DMXAA recognition (Figure 3E). The S162A/
Q266I double substitution was able to induce DMXAA responsiveness in all hSTING alleles, whereas single substitutions
were only effective in hSTINGR232 and hSTINGH232. This was
further validated by titration of DMXAA concentrations (see Figure 3B for hSTINGR232 and Figures S4A and S4B for other
hSTING alleles), which showed a variable maximal IFN-b induction for different alleles but clear sigmoidal dose responses that
diverged by less than one order of magnitude in their EC50. Taken
together, these results indicate that the Q266I substitution renders hSTING responsive to DMXAA. Further, hSTING containing
Q266I and S162A substitutions lead to a DMXAA-dependent
IFN-b reporter response close to that observed for mSTING.
Crystal Structure of DMXAA Bound to hSTINGS162A/Q266I
To better understand how S162A and Q266I substitutions facilitate the IFN induction of hSTING by DMXAA, we solved the cocrystal complex of DMXAA with hSTINGS162A/Q266I (aa 155–341) at
2.42 A˚ resolution (X-ray statistics in Table S1). The complex
adopts the ‘‘closed’’ conformation, as reflected by the positioning of two DMXAA in the binding pocket and the formation
of the four-stranded, antiparallel b sheet lid over the bound li-
hSTINGS162A/G230I/Q266I Is More Sensitive to DMXAA than
mSTING in IFN-b Induction
We next tested whether combining the G230I lid substitution
with the binding-pocket substitutions S162A/Q266I would
further enhance hSTING sensitivity to DMXAA. We generated
the triple mutant of hSTING and tested its binding to DMXAA
by ITC, as well as IFN induction by DMXAA in transfected cells.
The ITC titration for hSTINGS162A/G230I/Q266I with added DMXAA
is plotted in Figure 4A and shows a higher binding affinity (KD:
0.99 mM) than that observed for hSTINGgroup2 (KD: 3.12 mM; Figure 1C) or hSTINGS162A/Q266I (KD: 1.99 mM; Figure 3C), indicating
that all three substitutions individually contribute to an increased
DMXAA sensitivity. This increase in affinity translates to synergistic functional effects, based on our luciferase reporter assays
in which hSTINGS162A/G230I/Q266I showed approximately two
Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors 1671
A
B
untreated
DMXAA
0.020
IFN -Gluc, RLU
IFN -Gluc, RLU
0.008
0.006
0.004
0.002
C
mSTING
hSTING
hSTINGS162A
hSTINGQ266I
hSTINGS162A/Q266I
0.015
0.010
hSTINGS162A/Q266I
0.005
6V
26
R
A
R
Q
R
G
R
Q
hSTINGR232
F
hSTINGS162A/Q266I
+
DMXAA
hSTINGHAQ
hSTINGAQ
hSTINGQ293
Q266I
AQ
Q293
0.0
S162A / Q266I
R
S162A
H
WT (H232)
G
Q266I
R
S162A / Q266I
H232
0.1
S162A
Q
WT (Q293)
R
Q266I
A
S162A / Q266I
H
S162A
HAQ
0.2
WT (AQ)
R
Q266I
R
S162A / Q266I
G
S162A
R
WT (HAQ)
R232
0.3
Q266I
293
untreated
DMXAA
S162A / Q266I
232
300
0.4
S162A
230
10
30 100
DMXAA (µM)
0.5
mSTING
71
3
Control Plasmid
Residue Positions
hSTING
Variants
IFN -Gluc, RLU
E
D
0.000
WT (R232)
C
on hS
tro TIN
lP G
la
hS
sm
TI
id
N
hS G S1
62
TI
A
N
GG
hS
16
6S
TI
N
hS G I2
35
TI
L
N
hS G Q
26
TI
6I
N
hS G Q2
66
TI
L
N
GQ
0.000
hSTINGH232
G
I235 I235
L170
Q266I
L170
S162A
Q266I
Q266I
S162A
I165 S162A S162A I165
Q266I
Figure 3. S162A and Q266I Substitutions Render hSTING Sensitive to DMXAA
(A) 293T cells were transfected with reporter constructs and the indicated hSTING variants. At 12 hr after transfection, cells were stimulated with 0.18 mM DMXAA
for another 12 hr, followed by luciferase assay. Shown are means of triplicates + SEM, representative of three independent experiments.
(B) DMXAA dose-response curves of 293T cells transfected with the indicated STING variants, illustrating one representative experiment out of three independent
experiments.
(C) ITC binding curve for complex formation between DMXAA bound to hSTINGS162A/Q266I (aa 140–379).
(D) Natural variants of hSTING (Yi et al., 2013). Five hSTING variants (shown in the left column) were studied in this work. The amino acid variations are shown in the
right column and colored in red.
(E) 293T cells were transfected and stimulated as in (A). Shown are the S162A and Q266I mutants of major hSTING alleles. WT denotes the respective allele in this
context. Shown are the means of triplicates + SEM, representative of three independent experiments.
(F) The 2.42 A˚ crystal structure of DMXAA bound to hSTINGS162A/Q266I (aa 155–341). The representations and color codes are the same as used in Figure 1E.
(G) Two alternate views of the hydrophobic interactions of DMXAA with hSTINGS162A/Q266I. The two bound DMXAA molecules are shown in space-filling
representation, with surrounded hydrophobic side chains shown in stick and dot representations.
See also Figures S1, S3, and S4.
orders of magnitude higher sensitivity than hSTINGG230I, as
well as an order of magnitude higher sensitivity than either
hSTINGS162A/Q266I or mSTING for IFN-b induction by DMXAA
(Figure 4B).
We also solved the crystal structure of DMXAA bound to
hSTINGS162A/G230I/Q266I (aa 155–341) at 2.37 A˚ resolution (X-ray
statistics in Table S1) in the ‘‘closed’’ conformation (Figure 4C).
As expected, we observed both the hydrophobic pocket
1672 Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors
C
6
F
hSTINGS162A/G230I/Q266I
+
DMXAA
IFNB1
A
4
2
CXCL10
0
18
hSTINGS162A/G230I/Q266I
G230I
0
30
R232
I235
10
I235
0
L170
L170
G
HA-hSTING
GAPDH
S162A S162A
I165
6I
2A
16
GS
/Q
26
0I
Q
A/
62
I165
/G
10 30 100 300
2A
3
DMXAA (µM)
16
0.0
0.1 0.3 1
6I
Q266I
Q266I
0I
0.2
20
hS
TI
N
E
0.4
5
L170
26
mSTING
hSTING
hSTINGS162A/G230I/Q266I
hSTINGS162A/Q266I
hSTINGG230I
10
23
IFN -Gluc, RLU
0.6
I235
IL-6
B
15
C
on
tro
h
S
l
h
hS hS ST TIN
T
I
TI IN NG G
N
G S G S1 G23
I171
6
0
20
R238
CCL5
Y240
D
12
Figure 4. Triple Substitution of G230I/S162A/Q266I Yields an hSTING Variant with Higher Affinity to DMXAA in Comparison to mSTING and
Robust Stimulation of Cytokines and Chemokines in Mouse Cells
(A) ITC binding curve for complex formation between DMXAA bound to hSTINGS162A/G230I/Q266I (aa 140–379).
(B) 293T cells were transfected with reporter constructs and the indicated STING variants. After 12 hr, cells were stimulated with increasing concentrations of
DMXAA for another 12 hr, followed by luciferase assay. Individual data points are means of triplicates ± SEM. Representative of three independent experiments.
(C) The 2.37 A˚ crystal structure of DMXAA bound to hSTINGS162A/G230I/Q266I (aa 155–341). The representations and color codes are the same as in Figure 1E.
(D) Hydrophobic interactions of G230I substitution in the complex of DMXAA bound to hSTINGS162A/G230I/Q266I. The representations and color codes are the same
as in Figure 1G.
(E) Hydrophobic interactions of DMXAA in the ligand binding pocket, with the same representations and color codes as in Figure 3G.
(F) BMDCs (1 3 106) generated from a STINGGt/Gt mouse were transduced with retroviruses expressing WT and various hSTING mutants. Two days after viral
infection, the BMDCs were treated with 50 mg/ml of DMXAA for 3 hr and cells were collected for real-time PCR to measure IFNB1, CXCL10, CCL5, and IL-6 mRNA
levels. Data shown are means ± SEM (n = 3), representative of two independent experiments.
(G) BMDCs were transduced with retroviruses expressing WT hSTING and various hSTING mutants. Cells were collected 2 days after retroviral infection and the
levels of hSTING were determined by western blot analysis.
See also Figure S4.
surrounding I230 (Figure 4D), which was the same as in the
hSTINGG230I-DMXAA complex (Figure 2D), and the hydrophobic
interactions within the DMXAA binding pocket (Figure 4E), which
were the same as in the hSTINGS162A/Q266I-DMXAA complex
(Figure 3G).
DMXAA Activates Type I IFN and Proinflammatory
Cytokine and Chemokine Production in
mSTING-Deficient BMDCs Reconstituted with
hSTING Substitutions
We previously showed that c[G(20 ,50 )pA(30 ,50 )p] and its linkage
analogs induce type I IFN and proinflammatory cytokine/chemokine production in a STING-dependent manner in bone-marrow-
derived macrophages (Gao et al., 2013b). To test whether various
hSTING substitutions can rescue the deficiency of type I IFN and
proinflammatory cytokine/chemokine production in response to
DMXAA in mSTING-deficient bone-marrow-derived dendritic
cells (BMDCs), we generated BMDCs from homozygous functional null STING mice (Goldenticket, STINGGt/Gt) (Sauer et al.,
2011). Retroviruses carrying WT hSTING or hSTING mutants
(hSTINGG230I, hSTINGS162A/Q266I, hSTINGS162A/G230I/Q266I, and
hSTINGS162A) were used to transduce these BMDCs. Although
WT hSTING did not induce the upregulation of IFN-b mRNA after
DMXAA treatment, we observed 2.6-, 3.1-, 4.2-, and 2.2-fold increases in IFN-b mRNA levels in BMDCs expressing
hSTINGG230I, hSTINGS162A/Q266I, hSTINGS162A/G230I/Q266I, and
Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors 1673
hSTINGS162A, respectively. Similar to the results obtained from
the luciferase reporter assays, we found that STINGGt/Gt BMDCs
expressing hSTINGS162A/G230I/Q266I had the highest IFN-b mRNA
induction after DMXAA treatment, corroborating that G230I
substitution and the pocket substitutions S162A/Q226I lead to
synergistic effects on hSTING sensitivity to DMXAA. We also
observed upregulation of CXCL10, CCL5, and IL-6 mRNAs in
BMDCs expressing various hSTING mutants (Figure 4F), with
hSTINGS162A/G230I/Q266I eliciting the strongest induction among
the four mutants after DMXAA treatment. We also collected supernatants at 18 hr after DMXAA treatment. At this time point,
hSTINGS162A/G230I/Q266I induced the highest level of CXCL10
production compared with the other hSTING substituents (Figure S4E). We confirmed hSTING protein expression in transduced cells by western blot analysis (Figure 4G).
DISCUSSION
Functional studies have demonstrated that DMXAA activates
mSTING, but not hSTING (Conlon et al., 2013; Kim et al., 2013).
DMXAA showed great promise in mouse cancer models, underscoring its potential for human application, notwithstanding
the outcome of a phase III clinical trial for non-small-cell lung carcinoma (Lara et al., 2011). Hence, it is important to recognize that
although DMXAA itself is no longer a viable drug, pharmacological modulation of STING remains an ideal therapeutic strategy
to pursue. For this purpose, we sought to define the molecular
basis underlying DMXAA species selectivity.
Given that DMXAA binding involves interactions with identical
amino acids in both mSTING and hSTING (Gao et al., 2013b),
nonconserved residues that do not participate in direct interaction with DMXAA must contribute to species-specific response
to DMXAA.
We identified a hydrophobic interaction between the
substituted I230 and the residues from both the lid region and
other parts of the protein in the hSTINGgroup2-DMXAA complex
(Figure 1G), a distinctive feature that was also found in the structure of the mSTING-DMXAA complex (Figure S2C). All residues
that form the hydrophobic pocket that contains I230 are
conserved in both hSTING and mSTING proteins. The gain of
function of hSTINGG230I and, inversely, the loss of function of
mSTINGI229G and mSTINGI229A in their ability to induce IFN
gene expression in response to DMXAA further confirmed the
critical role of this residue in species-specific recognition of
DMXAA (Figures 2A and 2B). Our crystal structure of the
hSTINGG230I-DMXAA complex also exhibited the active
‘‘closed’’ conformation (Figures 2C and 2D), further supporting
our conclusion that this single point substitution outside of the
binding pocket of hSTING critically modulates sensitivity to the
otherwise mouse-selective DMXAA ligand. Hydrophobic interactions could help facilitate formation of the lid region and other
parts of the protein, allowing mSTING to form the ‘‘closed’’
conformation more readily than hSTING in response to DMXAA.
In general, our structural studies indicate that mSTING is
induced more readily to assume the ‘‘closed’’ conformation
than hSTING in response to CDNs and their analogs. To overcome this intrinsic disadvantage of hSTING, we need to design
better-fitting DMXAA analogs to allow hSTING to overcome the
energy barrier when transitioning from an ‘‘open’’ to a ‘‘closed’’
state. To eventually enable the rational design of suitable
DMXAA modifications, we systematically introduced hSTING
substitutions within the binding pocket and tested their influence
on DMXAA-induced IFN-b production. Following this strategy
and guided by our cocrystal structures of STING substituents
with DMXAA, we identified two point substitutions within the
ligand binding pocket: S162A (reported previously; Gao et al.,
2013b) and Q266I, each of which strongly promotes DMXAA
recognition (Figure 3A). Our data suggest that modestly altered
DMXAA derivatives might be sufficient to bind and activate
hSTING. By introducing the above substitutions into the predominant hSTING alleles, we were able to restore a dose-dependent response to DMXAA in all cases (Figures S4A and S4B).
Strikingly, the S162A/G230I/Q266I triple substitution of
hSTING showed an order of magnitude higher activity than
mSTING (Figure 4B), indicating that all three substitutions are
required to confer a synergistic effect to DMXAA recognition.
hSTINGS162A/G230I/Q266I might therefore be used as a benchmark
hSTING synthetic allele in future drug development studies using
humanized mouse models.
In summary, we have provided a comprehensive structural,
biophysical, and functional analysis of DMXAA’s association
with select substitutions within hSTING. Our results highlight
the critical role of the lid residue at position 230 (229 in mSTING)
and unveil the structural basis for the mSTING selectivity of
DMXAA. Our structural and functional results also shed light on
strategies to restore an efficient hSTING response to DMXAA
based on the binding-pocket S162A and Q266I substitutions. Toward this goal, our current efforts to generate reciprocal DMXAA
derivatives with synthetic chemist collaborators are directed toward the synthesis of analogs containing polar groups (OH,
OCH3, F, Cl, and NO2) at the C1/C2 (S162A substitution) and
C7 (Q266I substitution) positions within the DMXAA ring so as
to form additional intermolecular hydrogen bonds, as well as to
replace the six-membered aromatic rings with their fivemembered counterparts. Given that two molecules of DMXAA
bind to the STING dimer, ongoing efforts are also being directed
toward the generation and evaluation of covalently linked
DMXAA dimers. In this context, it should be noted that 7methyl-XAA and 8-methyl-XAA, which involve the redistribution
of CH3 substitutions on the DMXAA scaffold, have been identified
as weak yet human-active and possibly also human-selective
DMXAA analogs (Tijono et al., 2013). Finally, we are conducting
an ongoing program to screen small-molecule libraries using
high-throughput approaches and mass-spectroscopy-based
detection assays to identify potential candidates with scaffolds
distinct from DMXAA that target hSTING. Imitating the effects
of these amino acid substitutions by rational design of reciprocal
DMXAA derivatives should lead to the development of humanactive STING agonists for antitumor, antiviral, and vaccine adjuvant applications.
EXPERIMENTAL PROCEDURES
Crystallization and Structure Determination
Crystals were grown using the sitting-drop vapor diffusion method, and
diffraction data were collected at synchrotron beamlines. All structures were
solved using the PHASER, COOT, and PHENIX programs.
1674 Cell Reports 8, 1668–1676, September 25, 2014 ª2014 The Authors
Isothermal Titration Calorimetry
The thermodynamic parameters of the binding reactions of STING with
cGAMP isomers and DMXAA were measured by ITC using a MicroCal
ITC200 calorimeter at 25 C.
Reconstitution of STING-Deficient Murine BMDCs with hSTING
BMDCs were generated by culturing bone marrow cells from STINGGt/Gt mice
in complete medium in the presence of GM-CSF for 10 days. BMDCs (1 3 106
cells/well) were infected with retroviruses expressing hSTING (WT and various
substitution mutants). At 48 hr after retroviral infection, cells were stimulated
with DMXAA.
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Luciferase Assay
HEK293T cells were reverse transfected with STING expression plasmids and
reporter constructs as described previously (Gao et al., 2013b). DMXAA was
added by culture medium replacement 12 hr later. Luciferase expression
was determined after another 12 hr.
For further details regarding the materials and methods used in this work,
see the Supplemental Experimental Procedures.
Diner, E.J., Burdette, D.L., Wilson, S.C., Monroe, K.M., Kellenberger, C.A.,
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ACCESSION NUMBERS
Gao, P., Ascano, M., Zillinger, T., Wang, W., Dai, P., Serganov, A.A., Gaffney,
B.L., Shuman, S., Jones, R.A., Deng, L., et al. (2013b). Structure-function analysis of STING activation by c[G(20 ,50 )pA(30 ,50 )p] and targeting by antiviral
DMXAA. Cell 154, 748–762.
The following coordinates have been deposited to the Protein Data Bank:
DMXAA-hSTINGgroup2 (4QXO), DMXAA-hSTINGG230I (4QXP), DMXAAhSTINGS162A/Q266I (4QXQ), and DMXAA-hSTINGS162A/G230I/Q266I (4QXR).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and one table and can be found with this article online at http://
dx.doi.org/10.1016/j.celrep.2014.08.010.
AUTHOR CONTRIBUTIONS
The structural and in vitro binding assays were performed by P.G. under the
supervision of D.J.P. The luciferase assays monitoring interferon induction
were performed by T.Z. under the supervision of W.B. and G.H. The assays
describing DMXAA stimulation of BMDCs were performed by W.W. and P.D.
under the supervision of L.D. All authors participated in the writing of the paper
and agree with the contents.
ACKNOWLEDGMENTS
We thank the synchrotron beamline staffs of the Brookhaven National Laboratory and Argonne National Laboratory for their assistance. We thank Dr. Russell Vance (University of California, Berkeley) for providing us with the STINGGt/
Gt
mice. We thank Cristian Serna-Tamayo for excellent technical assistance.
D.J.P. is supported by grants from the Abby Rockefeller Mauze Trust, the Maloris Foundation, and the STARR Foundation. T.T. is supported by the HHMI.
L.D. is supported by NIH R56 AI095692-01. W.B. and G.H. are members of the
DFG Excellence Cluster ImmunoSensation and the German Centre for Infection Research (DZIF). W.B. and G.H. are supported by DFG grants SFB670
and SFB704. P.G. is supported by an Irvington Fellowship from the Cancer
Research Institute. Support for this project was provided by a grant from the
Robertson Foundation.
Received: June 25, 2014
Revised: July 14, 2014
Accepted: August 6, 2014
Published: September 4, 2014
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