Ligand-induced Ordering of the C-terminal Tail Primes STING for Phosphorylation by TBK1

• The binding of the potent ligand cGAMP to STING induces local structural ordering in the flexible C-terminal tail of STING.
• Site-directed mutagenesis studies, designed based on this observation, elucidated residues essential for STING function.
• The local structure formation in the CTT was shown to be essential for the induction of IFN-β production.

The innate immune protein Stimulator of interferon genes (STING) promotes the induction of interferon beta (IFN-β) production via the phosphorylation of its C-terminal tail (CTT) by TANK-binding kinase 1 (TBK1). Potent ligands of STING are, therefore, promising candidates for novel anti-cancer drugs or vaccine adjuvants. However, the intrinsically flexible CTT poses serious problems in in silico drug discovery. Here, we performed molecular dynamics simulations of the STING fragment containing the CTT in ligand-bound and unbound forms and observed that the binding of a potent ligand cyclic GMP-AMP (cGAMP) induced a local structure in the CTT, reminiscent of the known structure of a TBK1 substrate. The subsequent molecular biological experiments confirmed the observed dynamics of the CTT and identified essential residues for the activation of the IFN-β promoter, leading us to propose a new mechanism of STING activation.