Uncovering the Mechanism of Forkhead-Associated Domain-Mediated TIFA Oligomerization That Plays a Central Role in Immune Responses

Abstract

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

TRAF-interacting protein with forkhead-associated domain (TIFA) transduces DNA damage–induced activation of NF-κB

“…This observation was consistent with the binding preference of the FHA domain for phosphorylated threonine over phosphorylated serine (11,12,28). Indeed, we found threonine 9 of TIFA was phosphorylated in response to DNA insults, and the FHA-pThr interaction was crucial for TIFA-mediated NF-B activation in genotoxic conditions as its roles in other inflammatory pathways (14,23,29). Benefits of chromatin enrichment of TIFA for NF-B activation could be easily grasped with the fact that oligomerization is a prevailing mechanism for ubiquitination-based efficient assembly of IKK complex (4,5).…”

“…This point was further supported by the marginal induction of NEMO ubiquitination by FHA domain-deleted TIFA following ETO treatment ( Fig. 5d), in light of the critical involvement of the FHA domain to recognize pThr-9 for TIFA oligomerization in NF-B activation (13, 14,23). Interestingly, with the use of the nuclear lysate prepared from ETO-treated cells, we found the cells transfected with the TIFA-T9A mutant showed decreased DNA damage-elicited NEMO ubiquitination, especially its poly-ubiquitination forms at higher molecular weight, compared with the result from WT TIFA-transfected cells, whereas the ubiquitination pattern of cytoplasmic NEMO was nearly unaffected (Fig.…”

TIFA as a crucial mediator for NLRP3 inflammasome

“…Significantly, TIFA oligomerization seems to predominantly depend on pT9 rather than FHA or TRAF6 binding sites (i.e., Ser66 and Glu178, respectively) (10). The recently reported TIFA crystal structure supports such higher-order assembly of TIFA (11). Because TIFA activates signal 1 via TRAF6 oligomerization and signal 2 via the assembly of the inflammasome component proteins, phosphorylation of TIFA T9 would be essential for the innate immune response.…”

“…The crystal structure of TIFA indicates that the pT9-FHA interaction can occur only between different sets of dimers. Such homophilic TIFA interaction leads to the formation of the higherorder TIFA oligomer (11). This mode of TIFA oligomerization is required for TIFA interaction with TRAF6 and the ensuing activation of NF-κB (10,11).…”

“…Such homophilic TIFA interaction leads to the formation of the higherorder TIFA oligomer (11). This mode of TIFA oligomerization is required for TIFA interaction with TRAF6 and the ensuing activation of NF-κB (10,11). By using a genome-wide RNA interference screening, Gaudet et al recently showed that the pT9-FHA interaction is also essential for the innate immune response in the host induced by heptose-1,7-bisphosphate, a PAMP derived from Gramnegative bacteria (12).…”