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 MassachusettsWorcester (not Amherst)

Immediate and Delayed Anti-Viral Responses

IFN- IFN-

IFNAR

Jak1Tyk2

Stat2

Stat1

IRF-9 (ISGF3)

other cytokines,anti-viral genes

IFN-/

virus

IRF3

IRF3IRF3

IRF3

IRF3

IRF3

IFN- genes

PP

IFN- gene

IRF3

IRF3IRF3

P

IRF3P

TLR7,TLR8

ssRNA

MyD88

TBK1

IKK

IRF7

IRAK4

IRAK1TRAF6

Ub

TABs

1

TAK1

23

IKK

IKK

IKK

P

MAPKsNF-B

IBs

P

Ub

P

IBs

26Sproteasome

NF-BIRF7 ATF2/c-Jun

IFN

IRF5NF-B

Inflammatorycytokines

Cytoplasm

Endosome

Nucleus

IFNs

IRF7

TLR9

dsDNA

virus

MyD88

TRAF6Ub

IRF5Ub

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

P P

Cytoplasm

Nucleus

C

N

CBP

P P

DD

P P

Serine PO4 sites

B.Y. Qin, et al. K. Lin (2003) Nat. Struct. Biol. 10, 913 -921K. Takahashi, et al. F. Inagaki (2003) Nat. Struct. Biol. 10, 922-927

Domain structure of human IRF-3

110 427

NES

DBD1

NLS

200

IAD

IAD173 427

AUD

RVGGASSLENTVDLHISNSHPLSLTS

380

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 173-427)

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, 1269-1277

IRF-3 (residues 173-394)

CBP (2067-2112)

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

110 427

NES

DBD1

NLS

200

405

IAD

IAD173 427

AUD

Domain structure of Human IRF-3 and IRF-5

RVGGASSLENTVDLHISNSHPLSLTS

SGELSWSADSIRLQISNPDIKDRMV

NES

DBD

NLS

IAD

380

380

IRF-3

IRF-3 transactivation domain construct

IRF-5 (variant 4)

IRF-5 transactivation domain construct

IAD

222 467

NLS

421 455

421 4672331401

Interactions of CBP (2067-2112) with IRF-5 (222-467) and phosphomimetic mutants based on ITC data

Complex Kd Change in affinity

CBP – IRF-5 1.64M 1.0 foldCBP – IRF-5 (S427D) 0.96M 1.7 foldCBP – IRF-5 (S425D) 0.71M 2.3 foldCBP – IRF-5 (S436D) 0.67M 2.4 foldCBP – IRF-5 (S430D) 0.56M 2.9 fold

0

200

400

600

mAU(280 nm)

12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0

Volume (ml)

IRF-5

IRF-5 + CBP

CBP 200

400

600

800

mAU (280 nm)13.0 14.0 15.0 16.0 17.0

Volume (ml)

0

250 µM

IRF-5 WT

100 µM

50 µM

450 µM

0

200

400

600

800

mAU (280 nm)

13.0 14.0 15.0 16.0 17.0Volume (ml)

IRF-5 S430D450 µM

250 µM

100 µM

50 µM

mAU (280 nm)

0

200

400

600

12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Volume (ml)

IRF-5 S430DIRF-5 S430D + CBP

CBP

Size exclusion chromatography to investigate oligomerization of IRF-5 (222-467) and IRF-5 S430D

Monomer

Dimer

IRF-3 complex with CBP

C

N

IRF-5 dimeric subunit

C

N

Helix 2

Hel

ix 5

Helix 4

Helix 3 Helix 1

IRF-3 autoinhibited monomer

C

N

Helix 5

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

IRF-5 (222-467) S430D Dimer

Hel

ix 5

Hel

ix 5

N

N

C

C

IRF-5 (222-467) S430D Dimer

Hel

ix 5

Hel

ix 5

N

N

C

C

Helix 5 plays key alternate roles in IRF autoinhibition and dimerization.

IRF-5 dimerIRF-3 monomer

R353

I431’

L433’

I435’

S430’(D) S427’S425

S436’

K449’

V445’

D442’

R328

L403

Y303

L307

V310D312

F279

Helix 5

Hel

ix 2

Hel

ix 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

V310D312

F279

Helix 5

Hel

ix 2

Hel

ix 4

V391

L393

S396

I395 Hel

ix 1

Hel

ix 4

IRF-5 Dimer IRF-3 Monomer

R353

I431’

L433’

I435’

S430’(D) S427’S425

S436’

K449’

V445’

D442’

R328

L403

Y303

L307

V310D312

F279

Helix 5

Hel

ix 2

Hel

ix 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

0

100

200

300

400

500

600

mAU (280 nm)

13 14 15 16 17 18 19 20

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

luce

rfer

ase

(F

old

In

du

ctio

n)

I431’

L433’

I435’

S430’(D) S427’S425

S436’

K449’

V445’

D442’

R353

R328

L403

Y303

L307

V310D312

F279

Helix 5H

elix

2

(homologous IRF3 residue number for absolutely conserved residues)(L362)

(R285)

Hel

ix 5

R328

D442’

R353

S436’

Helix 5

IRF3-S386D/S396D/L362D & CBP IRF3-S386D/S396D/R285E & CBP IRF3-S386D/396D & CBP

0

100

200

300

400

mAU (280 nm)

Volume (ml)

12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0

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 activationIF

N

luc

erf

era

se

(F

old

In

du

cti

on)

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, 1111-1123

(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)

Phosphorylation

IRF activation, dimerization and CBP binding

C-term

(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)

Phosphorylation

IRF activation, dimerization and CBP binding

P

PP

PP

Hel

ix 5

Helix 5DBD

CBP

bindingsite

DBDCBP

bindingsite

DBD

DBD

Helix 5

Helix 5

Nucleus

Cytoplasm

DBD

DB

D

PP

PP

Hel

ix 5

CBP

binding

site

DBDCBP

binding

site

Helix 5

PP

PP

Hel

ix 5

Helix 5DBD

CBP

bindingsite

DBDCBP

bindingsite

CBP

University of Massachusetts, Worcester

Dept. of Biochemistry and Molecular Pharmacology

Kai Lin

Weijun ChenSuvana Lam

Hema SrinathBrendan Hilbert

Celia Schiffer

Department of Medicine

Kate FitzgeraldZhaozhao 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, 1111-1123

Binding of four N-terminal DNA Binding Domains of IRF-3 to promoter as part of IFN- enhanceosome