in a STING dimer. In Model 2, binding of a
monomeric MSA-2 molecule to one STING sub-
unit alters the MSA-2 affinity of the unoccupied
STING subunit. In Model 3, MSA-2 exists as
monomers and dimers in equilibrium, but only
dimers can bind STING. These mechanistic
models were probed experimentally.
Saturation binding of [^3 H]-MSA-2 to hSTING-
WT yielded a Hill coefficient >1 (n~ 1.6; solid
line in Fig. 5D) and a concave downward
Scatchard plot (Fig. 5D). Furthermore, homol-
ogous radioligand competition experiments
exhibited a bell-shaped relationship between
specific [^3 H]-MSA-2 binding to hSTING-WT
and unlabeled MSA-2 concentration (Fig. 5E).
ThesebehaviorsareincompatiblewithModel
1, which would have yielded a Hill coefficient
of unity (dashed line in Fig. 5D), a linear
Scatchard plot, and a homologous competi-
tion curve that decreased monotonically with
increasing unlabeled [MSA-2]. Furthermore,
Model 1 failed to account for the kinetics of
MSA-2 interaction with human and mouse
STING observed by surface plasmon resonance
(SPR) (Fig. 5F and fig. S4). On the basis of these
Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 4of10
Fig. 5. Mode of MSA-2–STING interaction.
(A) Structure (PDB ID 6UKM) of hSTING-HAQ dimer
(blue) with MSA-2 (green spheres). (B)Close-up
of bound MSA-2. NMR-relevant protons (white) are
annotated. (C) Three models in which L, L 2 ,and
R denote monomeric MSA-2 (dark green triangles),
dimeric MSA-2, and dimeric STING, respectively.
Visual depiction of Model 3 and equations are used
throughout this figure. (D) Saturation binding of
[^3 H]-MSA-2 (light green triangles) to full-length,
membrane-anchored hSTING-WT (graphic).n,Hill
coefficient;a, total membrane protein. (Inset)
Corresponding Scatchard plot. (E) Homologous
competition experiment with [^3 H]-MSA-2 (fixed),
hSTING-WT, and unlabeled MSA-2 fitted with an
equation based on Model 3 (solid line,KD).KD2and
[L] 50 were calculated from Eqs. 1 and 2 in (C) with
KD1 = 18 mM. Visualizations and graphics (inset)
depict predominant equilibria and STING complexes,
respectively, per concentration. CPM, counts per
minute. Error bars indicate SD. (F) Binding kinetics
(SPR) of MSA-2 to hSTING-WT (green trace).KD2was
determined by fitting (1:1, black line) using dimeric
[MSA-2] (L 2 ) calculated from Eq. 3 in (C). RU,
resonance units. (G) Proton NMR of MSA-2 titration to
estimateKD1 (18 mM; fig. S6). Proton annotations
correspond to those in (B).d, chemical shift. (H)Same
as (G) for compound 2 (inverted triangles).
(I) Simultaneous observation of hSTING-WT cytosolic
domain (graphic)–bound MSA-2 (triangles, titration)
and compound 2 (inverted triangles), monitored
by affinity selection mass spectrometry (MS) and
normalized independently (fig. S7 and text S2). Error
bars indicate SD. (J)IFN-bproduction in response
to MSA-2 titrations (alone; green) at multiple concen-
trations of compound 2 (orange) fit to a four-
parameter model (lines).
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