Protein–Ligand Interactions in the STING Binding Site Probed by Rationally Designed Single-Point Mutations: Experiment and Theory

STING
protein (stimulator of interferon genes) plays an important
role in the innate immune system. A number of potent compounds regulating
its activity have been reported, mostly derivatives of cyclic dinucleotides
(CDNs), natural STING agonists. Here, we aim to provide complementary
information to large-scale “ligand-profiling” studies
by probing the importance of STING–CDN protein–ligand
interactions on the protein side. We examined in detail six typical
CDNs each in complex with 13 rationally devised mutations in STING:
S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A,
T263S, R293Q, and G230A/R293Q. The mutations switch on and off various
types of protein–ligand interactions: π–π
stacking, hydrogen bonding, ionic pairing, and nonpolar contacts.
We correlated experimental data obtained by differential scanning
fluorimetry, X-ray crystallography, and isothermal titration calorimetry
with theoretical calculations. This enabled us to provide a mechanistic
interpretation of the differences in the binding of representative
CDNs to STING. We observed that the G230A mutation increased the thermal
stability of the protein–ligand complex, indicating an increased
level of ligand binding, whereas R238K and Y167F led to a complete
loss of stabilization (ligand binding). The effects of the other mutations
depended on the type of ligand (CDN) and varied, to some extent. A
very good correlation (R2 = 0.6) between
the experimental binding affinities and interaction energies computed
by quantum chemical methods enabled us to explain the effect of the
studied mutations in detail and evaluate specific interactions quantitatively.
Our work may inspire development of high-affinity ligands against
the common STING haplotypes by targeting the key (sometimes non-intuitive)
protein–ligand interactions.