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

Abstract

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.innate immunity | signaling | type I interferon | transcription factor | crystal structure

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

“…Moreover, the IRF3-STING (containing phosphorylated Ser 366 in CTT) complex may shed light on the structure of STING CTT which seems to be the most important conformational change upon CDNs binding. As expected, lately Pingwei Li's group determined the structure of IRF3-pSTING (residues 342-279) complex (169). Nevertheless, further work on the complex of IRF3-STING FL/ CTD (containing phosphorylated STING CTT) will be necessary to elucidate the detailed signaling.…”

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

“…Although formation of diS-bound dimer upon H2O2 and 2'3'-cGAMP stimulation was also impaired, the oxidation of Cys 148 was not directly demonstrated (60,61). Despite the fact that none of the available human ligand-bound STING structures allows to see the linker region containing Cys 148 in electronic density maps (29,(61)(62)(63)(64)(65)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91), it was proposed that Cys 148 is engaged in a ligand-inducible diS bond that stabilizes the uncrossed linker regions in the STING polymer (60,61). Our data, however, argues in favor of a different model in which Cys 148 oxidation is already present at basal levels.…”

Structural Basis for Shelterin Bridge Assembly

“…For biotinylated proteins, all target proteins were subcloned into a modified pET28a vector containing the Avi-6His-SUMO tag (Zhao et al, 2016). The biotinylated Avi-6his-SUMO-proteins were expressed in Rosetta BL21(DE3), which were co-transformed with the modified pET28a vector (Kanamycin) and the pBirAcm vector containing the BirA gene (Chloramphenicol).…”

“…( F ) and ( G ) Bio-Layer Interferometry (BLI) sensorgrams obtained using biosensors loaded with Biotin-SUMO-tagged Rap1 446-512 (Zhao et al, 2016), incubated with different concentrations of Poz1 (F) or Tpz1-Poz1 complex (G) used as analysts. Binding curves were fit globally to a 1:1 binding model to yield equilibrium dissociation constant (K d ), as well as association ( k on ) and dissociation ( k off ) rate constants for Poz1-Rap1 (R 2 =0.9778) (F), or Tpz1-Poz1 complex-Rap1 (R 2 =0.9996) (G) interactions.…”