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

Nipah virus G that precede its full activation. Nat Commun 8, 781,

doi:10.1038/s41467-017-00863-3 (2017).

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