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    • Molecular features of STING have been

    2018-10-23

    Molecular features of STING have been investigated by many research groups, and a number of amino hesperetin residues implicated for STING function have been identified. Those residues include phosphosites to induce the activation of the IFN-β promoter (Konno et al., 2013; Tanaka and Chen, 2012; Zhong et al., 2008); multiple studies using molecular biological techniques have identified both common and unique amino acids, such as S324, S326, S358 and S366 in human STING, to be phosphorylated on STING activation. In addition, genetic approaches with human SNP analysis or genome analysis of goldenticket mice have identified other amino acid residues important for ligand binding (Sauer et al., 2011). However, no correlation has been established so far between the IFN-β inducing activity of a ligand and its binding affinity (Gao et al., 2013). One possible explanation is the differences in the ability of these ligands to induce structural changes in STING. For example, changes in the dynamics of the CTT may lead to the phosphorylation of SER and THR residues by TBK1. In the current work, we adopted an interdisciplinary approach and predicted the dynamics of STING-CTT by in silico simulations, followed by the characterization of candidate residues by molecular biological techniques. We first constructed a structural model of the CTT in human STING and performed molecular dynamics (MD) simulations in ligand-bound and unbound forms. The simulation of cGAMP-bound human STING-CTT showed a behavior completely different from those in the c-di-GMP-bound and ligand-unbound forms. Site-directed mutagenesis studies, designed based on these observations, elucidated a phosphosite S354 in mouse (S355 in human) and three residues important for STING function, L362, L363 and M223 in mouse (L363, L364, and K224 in human, respectively), all of which have not been reported before. Our results suggest that a combination of computer simulations and molecular biological analysis is a powerful approach to investigating complex molecular features.
    Materials and Methods
    Results
    Discussions In both mouse and human STING, ligand binding causes a conformational change in the Lid, from open to close, and the natural ligands, cGAMP and c-di-GMP, with their binding affinity to STING, possess the ability to close the Lid (Huang et al., 2012; Zhang et al., 2013). However, several experimental studies showed that only cGAMP-bound human STING had sufficiently high IFN-β activity. In our simulation study of human STING, only the cGAMP-bound STING-CTT maintained the closed Lid conformation throughout the simulation (Fig. 1a), suggesting that the maintenance of the closed Lid conformation for a sustained period of time is required for the phosphorylation by TBK1 and the production of IFN-β. Only in the ligand-bound forms, the ends of the CTTs moved onto the Lid at an early stage of the simulations (during 28–29ns and 37–38ns in the cGAMP- and c-di-GMP-bound forms, respectively), and stayed on the Lid throughout the simulations (Fig. 1a), indicating that the conformational change in the Lid by ligand binding affects the behavior of the CTT. The conformational change in the Lid alters its electrostatic environment; it is highly positive in the ligand-unbound form, due to free K224, R232 and R238 pointing outward (Fig. 4a and see Materials and Methods for the calculation of the electrostatic potential), while it is lowly positive in the ligand-bound forms, because K224 is hydrogen-bonded to other Lid residues, and R232 and R238 interact with the ligand, making the positively charged side-chains in the ARG residues pointing inward (Fig. 4b). It suggests that changes in the electrostatic environments (weakened positive charges) on the Lid contributed to the movement of the CTT onto the Lid. The two ARGs above are conserved in mouse and human STING, while K224 in human corresponds to M223 in mouse. Therefore, the electrostatic property on the Lid in ligand-bound mouse STING is weaker than that in human STING (Fig. 4c). The site-directed mutagenesis study of K224M in human STING showed that this mutation enhanced IFN-β production in human STING (Fig. 3d). In general, IFN-β activity is higher in mouse than in human STING (Fig. 3d), suggesting that the weakening of the positive charges on the Lid leads to the enhanced IFN-β production. These observations support the hypothesis that the movement of the end of the CTT onto the Lid leads to phosphorylation by TBK1.