second-harmonic generation sensing of ligand- induced … · 2019-02-13 · second-harmonic...
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Second-Harmonic Generation Sensing of Ligand-Induced Conformational Changes in STING
David Shaya, Margaret Butko, Bason Clancy, Tracy YoungBiodesy, Inc., 170 Harbor Way #100, South San Francisco, CA-94080, USA
Second-Harmonic Generation
1. Butko, M. T., Moree, B., Mortensen, R. B. & Salafsky, J. Detection of Ligand-Induced
Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a
Supported Lipid Bilayer Interface. Anal Chem 88, 10482-10489,
doi:10.1021/acs.analchem.6b02498 (2016).
2. Ma, B. et al. ATP-Competitive MLKL Binders Have No Functional Impact on
Necroptosis. PLoS One 11, e0165983, doi:10.1371/journal.pone.0165983 (2016).
3. Moree, B. et al. Protein Conformational Changes Are Detected and Resolved Site
Specifically by Second-Harmonic Generation. Biophys J 109, 806-815,
doi:10.1016/j.bpj.2015.07.016 (2015).
4. Moree, B. et al. Small Molecules Detected by Second-Harmonic Generation
Modulate the Conformation of Monomeric alpha-Synuclein and Reduce Its Aggregation
in Cells. J Biol Chem 290, 27582-27593, doi:10.1074/jbc.M114.636027 (2015).
5. Spagnuolo, L. A. et al. Evaluating the Contribution of Transition-State
Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors. J
Am Chem Soc 139, 3417-3429, doi:10.1021/jacs.6b11148 (2017).
6. Wong, J. J. W. et al. Monomeric ephrinB2 binding induces allosteric changes in
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References Conclusion
SHG was successfully applied to detect and characterize binding interactions with STING:
1. Distinguished a wide range of CDN-induced conformational changes in STING
2. Sensitive to a wide range of CDN ligand affinities
3. The SHG data is consistent with orthogonal structural and biophysical techniques
SHG is uniquely situated to screen for therapeutically-relevant conformational changes in STING:
1. Fast optical readout enables screening of large libraries
2. Can resolve large and small conformational changes to reduce false negatives in screens from
alternative binding modes
3. Can differentiate between types of ligands based on binding mode to reduce false positives in
screens
4. Can resolve a wide range of interaction affinities, allowing for detection and characterization of
high and low affinity binders
BA
C
D
Ligand 1 Ligand 2
E
Stimulator of Interferon Genes (STING)
Stimulator of Interferon Genes (STING), an ER-resident membrane protein, acts as a cellular sensor
for cyclic dinucleotides (CDNs). Upon CDN binding, STING undergoes a large conformational change
to its active state, resulting in type I interferon-mediated innate immune response to pathogens.
Additionally, stimulation of the STING-cGAS pathway has been shown to activate T cells at tumor
sites, and a STING agonist could augment the effect of current checkpoint inhibitors in the tumor
microenvironment. Alternatively, inhibitors of STING signaling may be beneficial for the treatment of
autoinflammatory disease.
Detection of protein conformational changes by SHG (A) His-tagged protein is
conjugated with an SH-active dye (blue) and tethered onto a Ni-NTA lipid bilayer
(pink). Incoming light (red arrow) is directed at the dye generating a higher
frequency second-harmonic light (blue line). The SHG intensity is highly
dependent on the orientation of the dye with respect to the surface normal (Z-
axis). Ligand-induced conformational changes in the protein alters the net dye
orientation changing the SHG intensity measured by the Biodesy Delta™. (B) The
SHG signal depends on the average dye orientation and its distribution. (C) For
example: the apo conformation contains dye at position, S1 with an averageorientation angle Ɵ1, undergoes a conformational change to a new, ligand-bound
conformation, S2 with an average orientation angle Ɵ2. (D) The SHG intensity
will increase as the dye moves more parallel to the Z-axis (green ligand), and
the SHG intensity will decrease as the dye moves more perpendicular to the Z-
axis (blue ligand). (E) These differences are reported by the relative change in
SHG signal (Δ SHG).
SHG distinguishes CDN-induced
conformations of STING soluble CTD
The soluble CTD was expressed with a His tag, purified as a soluble protein, and
labeled at a native cysteine, Cys 148, with an SH-active dye. The protein was
tethered to a Ni-NTA bilayer, and SHG signal was measured upon injection ofthree distinct cyclic dinucleotides at saturation.
CDNs induced unique magnitude responses at saturation, suggesting unique ligand-bound conformations and consistent with previously published structural data.
CDN KD determination by SHG
SHG was used to measure concentration-response curves to determine the KD for these interactions.
SHG-determined KD for each CDN matches previously published values, confirming that SHG sensesbinding and reports conformational changes in STING in response to CDNs.
KD values were determined using the following non-linear model accounting for mass depletion:Buff
er
3'3
-cGAM
P
c-di-G
MP
2'3'
-cGAM
P
-20
0
20
40
60
80
SHG response to CDNs binding at sturation
D S
HG
(%
)
-10 -8 -6 -4-20
0
20
40
60
80
Log [compound] (M)
D S
HG
(%
)
3’3’-cGAMP
KD = 7.0 ± 0.8 mM
Receptor = 154 ± 43 nM
-10 -8 -6 -4-20
0
20
40
60
80
Log [compound] (M)
D S
HG
(%
)
c-di-GMP
KD = 2.8 ± 0.7 mM
Receptor = 154 ± 43 nM
-10 -8 -6 -4-20
0
20
40
60
80
Log [compound] (M)
D S
HG
(%
)
2’3’-cGAMP
KD = 24 ± 15 nM
Receptor = 154 ± 43 nM
Domain organization of human STING. Yellow star denotes native cysteine 148 that was labeled
with the SH-active dye and the round brackets represent the soluble CTD expressed and used in the
assay.
TM 2TM 1 TM 3 TM 4
Transmembrane domain CDN-binding domain
TBK1/IRF3
Binding
1-136 153-340 340-379
✶Cys 148
SHG construct = soluble C-terminal domain (CTD)
STING forms an obligate dimer arranged as a butterfly-shaped “open” structure in its apo state
(left). 3D structures reveal that upon binding of CNDs to the dimer interface, this protein undergoes
a conformational change to adopt the active closed conformation. Furthermore, different CDNs
induce distinct degrees of closing compared to the apo state. c-di-GMP (orange, middle) binding
induces modest closing mostly characterized by local conformational changes whereas 2’3’-cGAMP(blue, right) binding induces a considerable overall closing of the structure.
Structural plasticity of STING
Apo c-di-GMP 2’3’-cGAMP59.4 Å
PDB
accession
code 4F5W
PDB
accession
code 4F5Y
PDB
accession
Code 4LOH
59.3 Å 46.5 Å
0 2 4 6 8 10-20
0
20
40
60
80
Timecourse SHG response to CDNs
Time
D S
HG
(%
)
Buffer
2'3'-cGAMP
c-di-GMP
3'3 -cGAMP