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-/
virus
IFNAR
Tyk2
Jak1
Stat2
IRF3
IRF3
IRF3
IRF3
IRF3
Stat1
IRF3
IRF-9
(ISGF3)
P
P
IRF3
IRF3
P
IRF3
IFN- gene
P
IRF3
IFN- genes
IFN-
other cytokines,
anti-viral genes
IFN-
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.
Cytoplasm
virus
TLR7,
TLR8
ssRNA
dsDNA
TLR9
Endosome
MyD88
MyD88
IBs
IKK
Ub
IRAK4
IRAK1
Ub
TBK1
26S
proteasome
Ub
TRAF6
TRAF6
?
1
TABs
IKK
2
IKK
3
IKK
P
P
Ub
IBs
IRF5
IRF7
MAPKs
NF-B
IRF7 IRF5
IRF7
NF-B
ATF2/c-Jun
IRF5
P
NF-B
Nucleus
IFNs
IFN
Inflammatory
cytokines
P
IRFs are activated by phosphorylation in the C-terminal domain
P
C
P
P
N
Cytoplasm
Nucleus
P
CBP
P
P
P
IRF-3 acts as a molecular sentry for viral
infection in all cell types.
Serine PO4 sites
Domain structure of human IRF-3
380
RVGGASSLENTVDLHISNSHPLSLTS
110
1
200
DBD
NLS
380
427
IAD
AUD
NES
427
173
IAD
IRF-3 transactivation
domain construct
B.Y. Qin, et al. K. Lin (2003) Nat. Struct. Biol. 10, 913 -921
K. Takahashi, et al. F. Inagaki (2003) Nat. Struct. Biol. 10, 922-927
Structure of IRF-3 transactivation domain in complex with CBP supports the
hypothesis that the autoinhibitory region masks CBP binding site
CBP (2067-2112)
N
N
C
IRF-3 (residues 173-394)
IRF-3 (residues 173-427)
B.Y. Qin, et al. K. Lin (2005) Structure 13, 1269-1277
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
Domain structure of Human IRF-3 and IRF-5
405
380
RVGGASSLENTVDLHISNSHPLSLTS
IRF-3
110
1
200
DBD
NLS
380
427
IAD
AUD
NES
427
173
IRF-3 transactivation
domain construct
IAD
421
455
SGELSWSADSIRLQISNPDIKDRMV
IRF-5 (variant 4)
1
140
233
DBD
NLS
IRF-5 transactivation
domain construct
421 467
IAD
NLS
NES
222
467
IAD
Interactions of CBP (2067-2112) with IRF-5 (222-467) and
phosphomimetic mutants based on ITC data
Complex
CBP – IRF-5
CBP – IRF-5 (S427D)
CBP – IRF-5 (S425D)
CBP – IRF-5 (S436D)
CBP – IRF-5 (S430D)
Kd
Change in affinity
1.64mM
0.96mM
0.71mM
0.67mM
0.56mM
1.0 fold
1.7 fold
2.3 fold
2.4 fold
2.9 fold
Size exclusion chromatography to investigate
oligomerization of IRF-5 (222-467) and IRF-5 S430D
Monomer
450 µM
IRF-5
600
800
mAU (280 nm)
mAU(280 nm)
Dimer
IRF-5 + CBP
400
200
CBP
IRF-5 WT
600
250 µM
400
100 µM
200
50 µM
0
0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
13.0
19.0
14.0
Volume (ml)
15.0
Volume (ml)
16.0
17.0
450 µM
mAU (280 nm)
400
CBP
200
0
12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0
Volume (ml)
mAU (280 nm)
IRF-5 S430D
IRF-5 S430D + CBP
IRF-5 S430D
600
800
600
250 µM
400
100 µM
200
50 µM
0
13.0
14.0
15.0
Volume (ml)
16.0
17.0
C
N
N
Helix 1
C
C
N
Helix 2
IRF-3 autoinhibited monomer
IRF-5 dimeric subunit
IRF-3 complex with CBP
IRF-3 autoinhibited monomer IRF-5 dimeric subunit
IRF-3 complex with CBP
N
C
C
N
IRF-5 (222-467) S430D Dimer
N
C
C
N
IRF-5 (222-467) S430D Dimer
Helix 5 plays key alternate roles in IRF autoinhibition and dimerization.
IRF-3 monomer
IRF-5 dimer
S425
S430’(D) S427’
L403
I431’
Key interface residues
in the IRF-5 dimer
Y303
L433’
L307
R328
S436’
I435’
V310
D312
S436’
R353
R328
D442’
F279
D442’
V445’
K449’
R353
Helix 5
Helix 5
IRF-5 Dimer
IRF-3 Monomer
S425
S430’(D) S427’
L403
I431’
Y303
V391
L433’
L307
R328
L393
I435’
V310
D312
S436’
R353
I395
F279
D442’
V445’
K449’
Helix 5
S396
S425
S430’(D) S427’
L403
I431’
Key interface residues
in the IRF-5 dimer
Y303
L433’
L307
R328
S436’
I435’
V310
D312
S436’
R353
R328
D442’
F279
D442’
V445’
K449’
R353
Helix 5
Helix 5
Mutation of interface residues disrupt dimer
formation of IRF-5 in solution
Monomer
Dimer
600
IRF5-S430D & CBP
IRF5-S430D/V310D & CBP
IRF5-S430D/R328E & CBP
IRF5-S436D/R328E & CBP
IRF5-S430D/R353D & CBP
IRF5-S430D/D442R & CBP
mAU (280 nm)
500
400
300
200
CBP
100
0
13
14
15
16
17
Volume (ml)
18
19
20
Disruption of dimerization by mutation of
interface residues inhibits IRF-5 activation
IFN lucerferase (Fold Induction)
HEK293 Cells
S425
S430’(D) S427’
(homologous IRF3 residue
number for absolutely
conserved residues)
(L362)
L403
I431’
Y303
L433’
L307
(R285)
R328
I435’
V310
D312
S436’
S436’
R353
R328
F279
K449’
D442’
D442’
V445’
R353
Helix 5
Helix 5
Mutation of IRF-3 residues homologous to
IRF-5 dimeric interface residues disrupts
formation of the IRF-3 dimer in solution
Monomer
Dimer
mAU (280 nm)
400
IRF3-S386D/396D & CBP
IRF3-S386D/S396D/R285E & CBP
IRF3-S386D/S396D/L362D & CBP
300
CBP
200
100
0
12.0
13.0
14.0
15.0
16.0
17.0
Volume (ml)
18.0
19.0
20.0
IFN lucerferase (Fold Induction)
Disruption of IRF-3 dimerization inhibits its activation
HEK293 Cells
Published IRF-3 mutants reinterpreted in light of our
structure also support the crystallographically observed IRF5 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
IRF activation, dimerization and CBP binding
Phosphorylation
C-term
(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)
IRF activation, dimerization and CBP binding
Phosphorylation
(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)
P
P
P
P
P
Cytoplasm
Nucleus
CBP
P
P
P
P
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
Binding of four N-terminal DNA Binding Domains of
IRF-3 to promoter as part of IFN- enhanceosome
D. Panne, T. Maniatis & S.C. Harrison (2007) Cell 129, 1111-1123