The first direct high resolution structural information of an activated IRF reveals a dramatic phosphorylation-dependent structural rearrangement of the C-terminal region that triggers dimerization of IRF5. Thus, this work provides a structural basis for understanding how phosphorylation activates IRF family members.
Our mutagenesis studies support the observed dimer as representing the activated state of both IRF5 and IRF3. Published data on IRF3 mutants further supports the conclusion that IRF3 dimerizes similarly to IRF5. Qin et al.31
reported a triple mutation of loop L6 residues in which Val391, Leu393 and Ile395 (homologous with IRF5 residues Ile431, Leu433 and Ile435, respectively) were simultaneously mutated to Arg to disrupt the autoinhibitory contacts. As hypothesized, this triple mutant abolished the virus-dependent activation, but, unexpectedly, also interfered with IRF3 oligomerization. Such an interference with oligomerization of IRF3 is precisely the expected effect if the IRF3 dimer is similar to the IRF5 dimer, with these three residues in the interface. Additional published IRF3 mutants are also consistent with similar dimer formation to that of IRF5. These include double mutations, R211A R213A and K360A R361A, and triple mutations, R255A R262A H263A and R285A H288A H290A, that have been shown to inhibit viral-induced dimerization of IRF3 30,31
. In each mutant protein, one or more of the mutated residues is homologous with a residue in the IRF5 interface. Taken together, both the published data and the results presented here strongly support the crystallographically observed IRF5 dimer as representing the activated state of both IRF5 and IRF3.
The dramatic structural transitions observed for IRF5 appear to be a signature of the IRF family that is not shared with the evolutionarily related30,31
Smad proteins involved in TGF-β signaling. In both IRF and Smad proteins, phosphorylation of Ser/Thr residues in the C-terminal region triggers oligomerization. In Smad proteins, phosphorylation of serine residues in the C-terminal SSXS motif drives homo and hetero trimerization due to the direct binding of these phosphorylated serines in the trimer interface40
. In contrast, the phosphorylation sites in IRFs are more than 20 residues from the C-terminus. As a result, phosphorylation triggers much larger structural changes. Such structural changes can expose binding sites for other proteins, including CBP/p300, that contribute to signaling. In addition, the structural complexity of phosphorylation mediated signaling in IRFs suggests differential roles for individual sites: Ser425, Ser427 and Ser430 act primarily to destabilize the autoinhibited form, whereas Ser436 contributes to dimeric stability. These considerations suggest that conformational changes in IRF signaling may permit a complex differentiated biological response due to particular site specific phosphorylations.
Following phosphorylation IRF5 is translocated into the nucleus. Two potential nuclear localization signals (NLS) have been identified 10
. In contrast to IRF3 and IRF7, IRF5 can be detected in the nucleus of uninfected cells, which could result from the presence of two NLSs9,10
. Also unlike other IRFs, one putative NLS has been identified in the C-terminal transactivation domain. NLS signals are normally on unstructured surface loops that permit binding to importin-α (karyopherin-α)41-43
. The residues identified in the C-terminal domain as an NLS (398
) are in loop L5 that is intimately involved in the dimeric interface, which may mask them from binding importin-α. Thus the mutagenesis results on the putative NLS residues10
may need to be reevaluated, as such mutants likely disrupt dimerization which, in and of itself, could alter nuclear localization.
Dimerization of the IRF5 transactivation domain initiates the process leading to binding of full-length IRF5 to DNA. While no crystal structure of the N-terminal DNA binding domain (DBD) of IRF5 with DNA is available, structures of the well-conserved DBDs from several other IRF family members with DNA are available19-22
. The human IFN-β enhancer comprises binding sites for up to four IRF DBDs as well as activators NFκB and ATF-2/Jun. Pairs of DBDs that could reasonably be part of an IRF dimer have C-terminal α-carbon atoms separated by 42-49Å21,22
. These distances are consistent with the subunit arrangement for the IRF5 transactivation domain observed here, in which the N-terminal α-carbon atoms of the two IRF5 subunits are 59Å apart. Thus, the ~75 residue linker region between the N-terminal and C-terminal domains is not required to substantially alter the spacing between subunits for optimal DNA binding. However, this region must permit differences in the orientation of the two DBDs upon DNA binding since the two-fold symmetry relating the two C-terminal domains in the dimer is not present in the tandem bound DBDs (). As a result, dimerization of the transactivation domain brings the DBDs into only approximate position for binding DNA.
Figure 4 Schematic diagram for the activation of IRF family members, in which the interferon association domain (IAD) is shown as a crescent and the DNA Binding Domain (DBD) is shown as an oval behind the IAD. Unphosphorylated IRF proteins generally remain in (more ...)
IRF5 and IRF3 show substantially different responses to phosphorylation mimetics, with changes in affinity between CBP and IRF3 that are much more dramatic than those reported here for IRF5. In the case of IRF3, single phosphomimetic mutations increased affinity for CBP up to 19-fold and double mutations increased affinity up to 120-fold37
. In contrast, the largest change in affinity for binding CBP with IRF5 was only 2.9-fold (). These differences are consistent with earlier studies indicating that, unlike IRF3 and IRF7, an unphosphorylated autoinhibitory domain is not able to completely inhibit activation of IRF5 10
. Thus the phosphorylation dependent switch between autoinhibition and activation is more finely tuned in IRF5 than in IRF3.
The striking differences in sensitivity to activation between IRF3 and IRF5 likely represent different functional requirements that reflect distinct physiological roles of different IRFs. For instance, IRF3 is constitutively expressed in all cells, acting as a molecular sentry for viral infection. As such, it must be strongly autoinhibited, activated only in response to a clear signal. The C-terminal autoinhibitory/dimerization region plays a central role in activation. This region shows low sequence similarity (Supplementary Fig. 4
) among IRFs, including no absolutely conserved residues among IRF3 through IRF8. For IRF3, a large number of hydrophobic residues in the IAD likely contribute to its lower basal activity compared with IRF7 and IRF5 31
. An additional factor is the stability of the activated dimer. The IRF5 structure highlights a number of hydrophobic and ionic interactions that stabilize the dimer and may contribute to its greater basal activity. For instance, the ionic interaction between Arg353′ and Asp442 appears likely to be absent in IRF3 and IRF7 as the equivalent residues are Lys313 and Ser402 in IRF3 and Arg398 and Tyr488 in IRF7. In IRF3, K313A does not show a defect in virus-induced of dimer formation31
(the only reported non-defective IRF3 mutation of a residue homologous to an IRF5 dimeric interface residue). The absence of dimer stabilization by Lys313 may contribute to the tight autoinhibitory control of IRF3. Additionally, the hydrophobic surface formed by residues in the L1 loop that interacts with a portion of helix 5′ in IRF5 is absent in IRF3 and likely absent in IRF7 (Supplementary Fig. 4
), which could also weaken IRF3 and IRF7 dimers relative to the IRF5 dimer. Therefore the sensitivity of a given IRF family member is likely controlled through the balance between intramolecular autoinhibitory interactions and intermolecular dimeric interactions.
In conclusion, the structure of dimeric IRF5 reveals that phosphorylation-induced activation of IRF5 and other IRF family members involves a dramatic structural transition of the C-terminal autoinhibitory/dimerization region. Phosphorylation triggers unfolding of this region, allowing an extended conformation that binds to another IRF5 monomer. In doing so, the CBP binding site is exposed, providing a clear structural link between phosphorylation, dimerization and CBP/p300 binding (), key steps in transcriptional activation of IFN-β and associated genes.