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.T he presence of cytosolic DNA in mammalian cells is a danger signal that triggers the production of second messenger cGAMP by the DNA sensor protein cyclic GMP-AMP synthase (cGAS) (1, 2). cGAMP then activates the adaptor protein stimulator of IFN genes (STING, also known as TMEM173, MITA, ERIS, or MPYS) and induces the translocation of STING from the endoplasmic reticulum membrane to the Golgi, where it then recruits IκB kinase and TANK-binding kinase 1 (3, 4). The transcription factors nuclear factor κB and IFN regulatory factor 3 are subsequently activated, leading to the production of antiviral and proinflammatory cytokines, including type I interferons (IFNs). This cGAMP-mediated immune response is a fundamental defense mechanism against viral infection (5-7), making cGAMP and its structural derivatives potential vaccine adjuvants and immunotherapeutic agents against cancer (6,8).cGAMP is a hybrid cyclic dinucleotide that contains a guanosine unit and an adenosine unit joined by two phosphates in a macrocycle (Fig. 1). In contrast with the well-known bacterial signaling molecules cyclic di-GMP (c-di-GMP) and cyclic di-AMP (c-di-AMP) that contain a pair of (3′→5′)-phosphodiester linkages, the endogenous cGAMP molecule in mammalian cells contains unprecedented mixed phosphodiester linkages (9-12). Its two phosphate groups connect the two nucleosides from the 2′-and 5′ positions of guanosine and the 3′-and 5′ positions of adenosine. Denoted as 2′3′-cGAMP, this asymmetric ligand binds to the symmetric dimer of STING with an affinity significantly higher than its linkage isomers 2′2′-cGAMP, 3′2′-cGAMP, and 3′3′-cGAMP (SI Appendix, Table S1) (9). Notably, 3′3′-cGAMP has been found to be produced by certain bacteria (13, 14), whereas 2′2′-cGAMP and 3′2′-cGAMP have yet to be found in nature. Recent studies have revealed the molecular basis for the selective formation of the mixed 2′3′-phosphodiester linkages by cGAS (10, 14-18). However, the function of this linkage isomerism remained unclear and crystallographic studies of the STING/cGAMP complexes provided no insight (9, 19). Here we show that the preferred binding of 2′3′-cGAMP originates from its favorable free-ligand instead of protein-bound conformatio...

Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2′,3′ cGAMP Signaling

“…High affinity nvSTING–3′,3′ CDN interactions are made possible by a phenylalanine at the hSTING position 236, allowing a type of nucleobase stacking not possible with mammalian STING alleles. The uniquely compact regiochemical conformation of 2′,3′ cGAMP (Shi et al, 2015) allows it to target a more intermediately rotated state of STING where R238, a key residue involved in base-specific nvSTING–3′,3′ CDN, now completes the nucleobase stacking (Figure 6). It is possible that subsequent to the evolution of 2′,3′ cGAMP, F236 was lost to enhance discrimination of endogenous mammalian 2′,3′ cGAMP from bacterial 3′,3′ CDNs.…”

cGAS‐cGAMP‐STING: The three musketeers of cytosolic DNA sensing and signaling

“…Although it is well known that 2′3′‐cGAMP binds to STING with higher affinity than other linkage isomers like 2′2′‐cGAMP, 3′2′‐cGAMP and 3′3′‐cGAMP, structural studies clearly showed that cGAMPs bind to STING with indistinguishable modes. Recent data demonstrated that only inactive free‐ligand 2′3′‐cGAMP adopts an organized and favorable conformation which is more similar to the STING‐bound conformation owing to its specific G(2′,5′)pA phosphodiester linkage, thus it requires low entropy and enthalpy costs in converting into the active conformation . Besides natural 2′3′‐cGAMP, modified cGAMP linkages such as 2′3′‐cG s A s MP, a hydrolysis‐resistant bisphosphothioate analog of 2′3′‐cGAMP, was reported to retain even stronger STING agonist activity than 2′3′‐cGAMP .…”

Cellular and molecular regulation of innate inflammatory responses

“…RIG-I 11 and melanoma differentiation-associated protein 5 (MDA5) 12 can recognize viral dsRNA and recruit the CARD containing adaptor protein MAVS (also known as IPS-1, CARDIF or VISA), leading to IRF activation and the production of type I IFN. In addition to RLRs, a group of cytosolic DNA sensors such as cyclic GMP-AMP synthase (cGAS), [13][14][15] absent in melanoma 2 (AIM2), 16,17 DDX41, 18,19 Rad50, 20,21 LRRFIP1, 22 DNA-dependent activator of IRFs (DAI), 23 as well as various RNA sensors such as IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) 24 also play potent roles in inducing antiviral immune response, respectively via the adaptor protein stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS) or the MAVS pathway. cGAS, which was previously thought to recognize cytoplasmic dsDNA over 40 bp in a sequence-independent manner, is recently shown to recognize unpaired guanosines flanking short (12-20 bp) dsDNA (Y-form DNA) found in human immunodeficiency virus type 1 to induce type I IFN production.…”