Activation of the Interferon Regulatory Factors: Crystal Structure of Dimeric IRF-5 Bill Royer, Weijun Chen, Suvana Lam, Hema Srinath, Celia Schiffer, Kate Fitzgerald and Kai Lin University of Massachusetts Worcester (not Amherst)

Immediate and Delayed Anti-Viral Responses IFN-  IFN-  IFNAR Jak1 Tyk2 Stat2 Stat1 IRF-9 (ISGF3) other cytokines, anti-viral genes IFN-  /  virus IRF3 IFN-  genes P P IFN-  gene IRF3 P P

TLR7, TLR8 ssRNA MyD88 TBK1 IKK  IRF7 IRAK4 IRAK1 TRAF6 Ub TABs 1 TAK1 2 3 IKK  IKK  IKK  P MAPKs NF-  B I  Bs P Ub P I  Bs 26S proteasome NF-  B IRF7 ATF2/c-Jun IFN  IRF5 NF-  B Inflammatory cytokines Cytoplasm Endosome Nucleus IFN  s IRF7 TLR9 dsDNA virus MyD88 TRAF6 Ub IRF5 Ub IRF5 ? P Innate immunity is triggered by the recognition of “pathogen-associated molecular patterns” such as viral nucleic acids by Toll-like receptors (TLR) or cytoplasmic receptors.

IRFs are activated by phosphorylation in the C-terminal domain P PP Cytoplasm Nucleus C N CBP PP DD PP

Serine PO 4 sites B.Y. Qin, et al. K. Lin (2003) Nat. Struct. Biol. 10, K. Takahashi, et al. F. Inagaki (2003) Nat. Struct. Biol. 10, Domain structure of human IRF NES DBD 1 NLS 200 IAD AUD RVGGASSLENTVDLHISNSHPLSLTS 380 IRF-3 transactivation domain construct IRF-3 acts as a molecular sentry for viral infection in all cell types.

N C IRF-3 (residues ) Structure of IRF-3 transactivation domain in complex with CBP supports the hypothesis that the autoinhibitory region masks CBP binding site B.Y. Qin, et al. K. Lin (2005) Structure 13, IRF-3 (residues ) CBP ( ) N

IRF-5 The autoinhibition of IRF-5 is less tight than that for the ubiquitously expressed IRF-3. IRF-5 is activated by: viral expression type I interferon tumor suppressor p53 IRF-5 activates type I interferon inflammatory cytokines tumor suppressors Human mutations of IRF-5 have been implicated in systemic lupus erythematosis multiple sclerosis Sjogrens syndrome Inflamatory bowel disease IRF-5 k.o. mice show susceptibility to viral infection susceptibility to tumors

NES DBD 1 NLS IAD AUD Domain structure of Human IRF-3 and IRF-5 RVGGASSLENTVDLHISNSHPLSLTS SGELSWSADSIRLQISNPDIKDRMV NES DBD NLS IAD 380 IRF-3 IRF-3 transactivation domain construct IRF-5 (variant 4) IRF-5 transactivation domain construct IAD NLS

Interactions of CBP ( ) with IRF-5 ( ) and phosphomimetic mutants based on ITC data Complex K d Change in affinity CBP – IRF  M1.0 fold CBP – IRF-5 (S427D)0.96  M1.7 fold CBP – IRF-5 (S425D)0.71  M2.3 fold CBP – IRF-5 (S436D)0.67  M2.4 fold CBP – IRF-5 (S430D)0.56  M2.9 fold

mAU(280 nm) Volume (ml) IRF-5 IRF-5 + CBP CBP mAU (280 nm) Volume (ml) µM IRF-5 WT 100 µM 50 µM 450 µM mAU (280 nm) Volume (ml) IRF-5 S430D 450 µM 250 µM 100 µM 50 µM mAU (280 nm) Volume (ml) IRF-5 S430D IRF-5 S430D + CBP CBP Size exclusion chromatography to investigate oligomerization of IRF-5 ( ) and IRF-5 S430D Monomer Dimer

IRF-3 complex with CBP C N IRF-5 dimeric subunit C N Helix 2 Helix 5 Helix 4 Helix 3 Helix 1 IRF-3 autoinhibited monomer C N Helix 5

IRF-5 dimeric subunit IRF-3 autoinhibited monomer IRF-3 complex with CBP

IRF-5 ( ) S430D Dimer Helix 5 N N C C

IRF-5 ( ) S430D Dimer Helix 5 N N C C

Helix 5 plays key alternate roles in IRF autoinhibition and dimerization. IRF-5 dimer IRF-3 monomer

R353 I431’ L433’ I435’ S430’(D) S427’ S425 S436’ K449’ V445’ D442’ R328 L403 Y303 L307 V310 D312 F279 Helix 5 Helix 2 Helix 4 Key interface residues in the IRF-5 dimer R328 D442’ R353 S436’ Helix 5

R353 I431’ L433’ I435’ S430’(D) S427’ S425 S436’ K449’ V445’ D442’ R328 L403 Y303 L307 V310 D312 F279 Helix 5 Helix 2 Helix 4 V391 L393 S396 I395 Helix 1 Helix 4 IRF-5 DimerIRF-3 Monomer

R353 I431’ L433’ I435’ S430’(D) S427’ S425 S436’ K449’ V445’ D442’ R328 L403 Y303 L307 V310 D312 F279 Helix 5 Helix 2 Helix 4 Key interface residues in the IRF-5 dimer R328 D442’ R353 S436’ Helix 5

IRF5-S430D & CBP IRF5-S430D/R353D & CBP IRF5-S430D/D442R & CBP IRF5-S430D/V310D & CBP IRF5-S430D/R328E & CBP IRF5-S436D/R328E & CBP mAU (280 nm) Volume (ml) Dimer Monomer CBP Mutation of interface residues disrupt dimer formation of IRF-5 in solution

Disruption of dimerization by mutation of interface residues inhibits IRF-5 activation HEK293 Cells IFN  lucerferase (Fold Induction)

I431’ L433’ I435’ S430’(D) S427’ S425 S436’ K449’ V445’ D442’ R353 R328 L403 Y303 L307 V310 D312 F279 Helix 5 Helix 2 (homologous IRF3 residue number for absolutely conserved residues) (L362) (R285) Helix 5 R328 D442’ R353 S436’ Helix 5

IRF3-S386D/S396D/L362D & CBP IRF3-S386D/S396D/R285E & CBP IRF3-S386D/396D & CBP mAU (280 nm) Volume (ml) Dimer Monomer CBP Mutation of IRF-3 residues homologous to IRF-5 dimeric interface residues disrupts formation of the IRF-3 dimer in solution

Disruption of IRF-3 dimerization inhibits its activation IFN  lucerferase (Fold Induction ) HEK293 Cells Published IRF-3 mutants reinterpreted in light of our structure also support the crystallographically observed IRF- 5 dimer as representing the active state of IRF-3

Relationship of the IRF-5 transactivation domain dimer with IRF-3 DNA binding Domains D. Panne, T. Maniatis & S.C. Harrison (2007) Cell 129,

(Morphing CNS script from the Yale Morph Server, Phosphorylation IRF activation, dimerization and CBP binding C-term

(Morphing CNS script from the Yale Morph Server, Phosphorylation IRF activation, dimerization and CBP binding

P P P P P Helix 5 DBD CBP binding site DBD CBP binding site DBD Helix 5 Nucleus Cytoplasm DBD P P P P Helix 5 CBP binding site DBD CBP binding site Helix 5 P P P P DBD CBP binding site DBD CBP binding site CBP

University of Massachusetts, Worcester Dept. of Biochemistry and Molecular Pharmacology Kai Lin Weijun Chen Suvana Lam Hema Srinath Brendan Hilbert Celia Schiffer Department of Medicine Kate Fitzgerald Zhaozhao Jiang $ - NIH

Sequence alignment of the C-terminal transactivation domains of human IRF family members

The IFN  Enhanceosome IRF3 IRF7

D. Panne, T. Maniatis & S.C. Harrison (2007) Cell 129, Binding of four N-terminal DNA Binding Domains of IRF-3 to promoter as part of IFN-  enhanceosome