Ligand Strain and Its Conformational Complexity Is a Major Factor in the Binding of Cyclic Dinucleotides to STING Protein

Abstract

STING (stimulator of interferon genes) is a key regulator of innate immunity that has recently been recognized as a promising drug target. STING is activated by cyclic dinucleotides (CDNs) which eventually leads to expression of type I interferons and other cytokines. Factors underlying the affinity of various CDN analogues are poorly understood. Herein, we correlate structural biology, isothermal calorimetry (ITC) and computational modeling to elucidate factors contributing to binding of six CDNs-three pairs of natural (ribo) and fluorinated (2'-fluororibo) 3',3'-CDNs. X-ray structural analyses of six {STING:CDN} complexes did not offer any explanation for the different affinities of the studied ligands. ITC showed entropy/enthalpy compensation up to 25 kcal mol^-1 for this set of similar ligands. The higher affinities of fluorinated analogues are explained with help of computational methods by smaller loss of entropy upon binding and by smaller strain (free) energy.

Keywords: conformational analysis; cyclic dinucleotides; entropy; quantum chemistry; strain energy.

Figure 1

C‐terminal CDN binding domain of human wild‐type STING in open and closed conformations. A) Unliganded STING in an open conformation (PDB entry 4EMU). ^[31] B) STING in complex with its natural agonist 2′,3′‐cGAMP in a closed conformation (PDB entry 4KSY). ^[32] Displayed distances are measured between Cα atoms of Gln184 of each monomer.

Figure 2

Cyclic dinucleotides studied in this work: A) cGAMP, B) F₂ cGAMP, C) cdi‐GMP, D) F₂ cdi‐GMP, E) cdi‐AMP, F) F₂ cdi‐AMP. ΔT _M values represent melting temperature differences of protein:ligand complexes in comparison with the unliganded STING.

Figure 3

Structural alignment of all structures of interest: A) STING in complex with cdi‐GMP (PDB entry 6S86), F₂ cdi‐GMP (PDB entry 6YDB), cGAMP (PDB entry 6YDZ), F₂ cGAMP (PDB entry 6YEA), cdi‐AMP (PDB entry 6Z15), or F₂ cdi‐AMP (PDB entry 6Z0Z). Complexes with natural ligands are represented in yellow, while their fluorinated analogues are in green. B) Structural alignment of studied ligands in bound conformations.

Figure 4

Thermodynamic characterization of STING:ligand complex forming process by ITC. Raw and integrated data of the titration of STING in Tris buffer with A) cGAMP, B) F₂ cGAMP, C) cdi‐GMP, D) F₂ cdi‐GMP. The c‐values for titrations: STING protein with ligand cGAMP (c=6), F₂ cGAMP (c=480), cdi‐GMP (c=5), and F₂ cdi‐GMP (c=33).

Figure 5

Binding modes of ligands of interest and their interaction with STING: A) cGAMP vs. F₂ cGAMP; B) cdi‐AMP vs. F₂ cdi‐AMP; C) cdi‐GMP and F₂ cdi‐GMP (note that only these two form additional three H‐bonds with Ser241).

Figure 6

Thermodynamic cycle used to analyze the binding of CDNs to STING. The direct path (bottom) corresponds to the values measured by ITC. For computational analysis, we split this process into three steps. Step 1 represents restricting a conformational ensemble of free ligand in solvent to a single structure—a global minimum. Step 2 represents changing the conformation from global minimum to a bound‐like structure. Step 3 represents replacing the solvent environment with a cluster model of protein–ligand complex. Double difference values are defined as ΔΔX=ΔX(natural)−ΔX(di‐F).