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

Abstract

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,

How fragile we are. Influence of STimulator of INterferon Genes, STING, variants on pathogen recognition and immune response efficiency

“…This observation is coherent with recent Isothermal Titration Calorimetry (ITC) results, which suggest that higher melting temperatures for the G230A cGAMP:STING complex might be related to a structural stabilization of the lid region, the ligand affinity of this variant being similar to the one observed for the WT. 46 In line with these observations, the binding free energy change upon G230A mutation predicted by Thermodynamic Integration calculations is of only 1.20±0.25 kcal/mol -see Table 1. 1.11 ± 0.65 G230A-R293Q 9.40 ± 0.68 On the contrary the strongly enhanced structuration of the lid region upon cGAMP binding is well evidenced by the flexibility profile -see Figure 4.…”

“…The R232 residues invoked in the literature 22 is instead located on the external face of the lid and stays relatively far from cGAMP all along the trajectory in the WT, but, as it will be discussed in the following, it shows a more important role in the A and AQ variants. Interestingly, the S162 residue of both monomer, which lies at the bottom of the cavity and whose mutation to T or A destabilizes the cGAMP:STING complex, 46 is also involved in the second sphere of interaction in our simulations.…”

“…Furthermore, hydrogen bonds between R238 and the ligand's phosphates and between the nucleobases and E260 and T263 are also emerging -see Figure 2. These residues have been previously proposed to take part in cGAMP recognition 46 and we also retrieve the previously-reported amino acids T267 in the second sphere of interaction together with Y163 and Y240 -see Figure S3. The R232 residues invoked in the literature 22 is instead located on the external face of the lid and stays relatively far from cGAMP all along the trajectory in the WT, but, as it will be discussed in the following, it shows a more important role in the A and AQ variants.…”

“…Upon binding of cGAMP, the two loops fold onto the ligand to structure into 'lids' at the edges of the binding cavity, as observed in previous experimental and theoretical studies. 19,20,22,46 Two arginines belonging to the loops, (R238 on each monomer) dive towards cGAMP and get stabilized by very strong cation-π interactions with the purine moieties of cGAMP -see Figure 2 and Globally the binding of cGAMP induces a very stable interaction network in the binding pocket. The latter involves, in addition to the R238 cation-π interactions already described, Y167 π-stacking with the cGAMP purines.…”

Enzymatic Synthesis of 3′–5′, 3′–5′ Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations

“…The assay was performed as described previously. , Briefly, the WT STING protein was diluted to a final concentration of 0.1 mg/mL in 100 mM Tris-HCl buffer at pH 7.4 containing 150 mM NaCl, 1:500 (v/v) SYPRO Orange, and 150 μM CDN or water. Solutions (20 μL) of the reaction mixtures were pipetted in triplicates into 96-well optical plates, and thermal denaturation of samples was performed on a real-time PCR cycler (LightCycler 480 Instrument II, Roche, Basel, Switzerland).…”