Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

Abstract

Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein-ligand interactions.

Keywords: STING; cGAMP; ligand conformation; phosphodiester linkage.

Fig. 1.

Structures of cyclic dinucleotides. cGAMP molecules are hybrid cyclic dinucleotides comprising a GMP and an AMP unit whereas c-di-GMP and c-di-AMP are homodimers of GMP and AMP, respectively. In contrast with the bacterial signaling molecules 3′3′-cGAMP, c-di-GMP, and c-di-AMP that contain a pair of homogeneous (3′→5′) phosphodiester linkages, the mammalian second messenger 2′3′-cGAMP contains unique G(2′→5′)/A(3′→5′) phosphodiester linkages.

Fig. 2.

Thermostabilization of STING by different cyclic dinucleotides. The melting curves of STING (10 μM) were recorded in the absence and presence of 100 μM cGAMP isomers or c-di-GMP. The T_m values (shown with 95% confidence intervals) were determined by fitting the data to a Boltzmann sigmoidal equation. Representative results of three independent experiments are shown.

Fig. 3.

Crystal structures of STING/2′3′-cGAMP complexes suggest that 2′3′-cGAMP and 3′2′-cGAMP would have the same binding interactions with STING. (A) 2′3′-cGAMP binds to STING in two alternative orientations with the two purine base groups placed at the same positions (PDB ID code 4KSY) (9). (B) Shuffling the guanine and adenine groups converts 2′3′-cGAMP into 3′2′-cGAMP.

Fig. 4.

STING binds to 2′3′-cGAMP and 3′2′-cGAMP with indistinguishable binding modes. (A) STING undergoes the same conformational change upon binding to 2′3′-cGAMP (green, PDB ID code 4KSY) (9) and 3′2′-cGAMP (yellow, PDB ID code 5BQX). (B) 2′3′-cGAMP (green) and 3′2′-cGAMP (yellow) bind to STING with nearly identical hydrogen bond networks.

Fig. 5.

Conformational analyses of the mammalian 2′3′-cGAMP molecule. 2′3′-cGAMP exists in (A) a set of organized conformations in solution, leading to (B) a closed equilibrium geometry that highly resembles (C) the STING-bound conformation.

Fig. 6.

Conformational analyses of the linkage isomer 3′2′-cGAMP. (A) 3′2′-cGAMP has high degrees of freedom as a free ligand with (B) an open equilibrium geometry that requires a large conformational change to bind to STING.

Fig. 7.

Conformational analyses of the linkage isomer 2′2′-cGAMP. The strong hydrogen bond network holds 2′2′-cGAMP in a closed conformation as a free ligand both (A) in gas phase and (B) in solution.