Structural Analysis of the STING Adaptor Protein Reveals a Hydrophobic Dimer Interface and Mode of Cyclic di-GMP Binding

Abstract

SUMMARY STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved AA153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of post-translational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than Ser358. This study provides a glimpse into the unique architecture of STING and sheds new light on the mechanism of c-di-GMP-mediated TBK1 signaling.

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

“…Mutagenesis studies identified key residues involved in binding of c di-GMP. In another recent study, the structure of c di-GMP bound STING (amino acids 139-343) accounted for the observation that dimerization of STING is essential for binding c di-GMP and not only maps the exact location but also provides information on molecular determinants for specificity in binding c di-GMP (Ouyang et al, 2012). This structural information coupled with mutagenesis and cell based studies firmly establishes STING as a bona fide protein that binds c di-GMP in vitro as well as in vivo and elicits IFN production.…”

“…4). Whether R231 is involved in binding or facilitating docking unliganded as well as liganded structures of STING (Ouyang et al, 2012). However, what is known and can be observed in the structure is that this loop sits right above the c di-GMP and a mutation of the R231A compromises the ability of STING to respond to c di-GMP, but not DNA.…”

“…A STING S358A mutation abolished the ability of STING to interact with TBK1 and the subsequent activation of IRF-E (Zhong et al, 2008). Evidence for an additional effector TBK1 binding site was unveiled in a clever STING-TBK1 GST pulldown study in presence of c di-GMP (Ouyang et al, 2012). Recruitment of TBK1…”

“…The crystal structure of the C-terminal domain of STING (STING CTD; AA 132-343) solved by using bacterially expressed protein, is made up of a single domain with a mixed α/β topology ( Fig. 5) (Ouyang et al, 2012). A central twisted sheet made up of 5 strands is surrounded by α-helices.…”

“…STING specifically bound c di-GMP and not ATP or GTP. This specificity is beautifully illustrated in the crystal structure of the binary complex of STING with c di-GMP, where the ligand binds in a trough just above the dimer interface (Ouyang et al, 2012). The c di-GMP binding pocket is constrained such that there is no space for accommodating additional phosphate groups of ATP or GTP.…”