Structural Insights into the Functions of TBK1 in Innate Antimicrobial Immunity

Abstract

SUMMARY Tank-binding kinase 1 (TBK1) is a serine/threonine protein kinase mediating innate antimicrobial immunity. TBK1 is involved in the signaling of TLRs, RLRs, and STING-mediated sensing of cytosolic DNA. Stimulation of these receptors results in the activation of TBK1, which phosphorylates interferon regulatory factor IRF-3. Phosphorylated IRF-3 translocates into the nucleus to initiate the transcription of the IFN-β gene. Here we show that TBK1 is activated by autophosphorylation at residue Ser172. Structures of TBK1 bound to two inhibitors showed that TBK1 has the IκB kinase fold with three distinct domains: the kinase domain, the ubiquitin like domain, and the scaffold and dimerization domain. However, the overall structures of TBK1 monomer and its dimer are different from IKKβ in the arrangements of the three domains and in dimer formation. Phosphorylation of IRF-3 by TBK1 in vitro results in its oligomerization, and phosphorylation of residue Ser386 plays a key role in IRF-3 activation.

Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins

“…The IRF-3/CBP dimer structure also explains how dozens of previously reported mutations affect IRF-3 activation (Fig. S5F) (27,29,(32)(33)(34). Most of these mutations are located at the tail-mediated IRF-3 dimer interface and play crucial roles in mediating IRF-3 dimerization (Fig.…”

“…Binding of the phospho-pLxIS motif to IRF-3 induces conformational changes that begin to reorganize key IRF-3 loops and uncover surfaces involved in dimerization, whereas colocalization of IRF-3 with TBK1 induces phosphorylation of the pLxIS motif in the IRF-3 tail. Phosphorylation of IRF-3 by TBK1 can occur in vitro but is relatively inefficient and requires high concentrations (34); colocalization of TBK1 and IRF-3 through recruitment via the adaptors increases local concentrations of both, thus facilitating the phosphorylation and dimerization of IRF-3. The scaffold mechanism for IRF-3 activation would protect effectively against the activation of IRF-3 in the cytosol by TBK1 until appropriate formation of the innate signaling machinery.…”

“…The SUMO tag was removed using a Ni 2+ -NTA column, and the target proteins or peptides in the flow through were further purified by gel-filtration chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare) eluted with 20 mM Tris·HCl (pH 7.5) and 150 mM NaCl. Human STING CTD (residues 155-379) and mouse TBK1 (residues 1-657) were expressed and purified as described previously (34). SUMO fusion of human CBP (residues 2065-2111) was cloned into the pET22b(+) vector.…”

Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation

“…Even though a mass spectrometry scan has suggested that Thr75 and Thr253 of IRF3 might be phosphorylated in cells (Shu et al 2013), the function implications of the phosphorylation and the kinases responsible for Thr75 or Thr253 modification have not been explored. The Thr75 residue is located in the DBD of IRF3; thus, it is not surprising that its phosphorylation disrupts DNA binding.…”

The Oncometabolite Fumarate Promotes Pseudohypoxia Through Noncanonical Activation of NF-κB Signaling

“…TBK1 is a member of the IKK-related kinases and is a known upstream mediator of non-canonical NF-B activation (29). TBK1 contains an autophosphorylation site at Ser 172 , which is indicative of TBK1 kinase activation (30). To determine the effects of fumarate on TBK1 activation, we treated DKO IKK␣/␤ MEFs with increasing concentrations of DEF.…”