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Regulation of biological activity through protein dimerization: Crystallographic analyses of primitive
hemoglobins and interferon regulatory factors
Bill RoyerUniversity of Massachusetts
Worcester (not Amherst)
Scapharca HbI Deoxy Lamprey HbV IRF-5 Dimer
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Sperm whale myoglobin(Structure by John Kendrew, illustration by Irving Geis)
Molluscan hemoglobin subunit
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Deuterostomes
ChordataEchinodermata
Echiura
MolluscaPr
otos
tom
es
Homo sapiens HbA(tetramer)
Petromyzon marinus HbV(deoxy dimer)
Caudina arenicola HbD(dimer)
Urechis caupo Hb(tetramer)
S. inaequivalvis HbI(dimer)
Scapharca inaequivalvis HbII (tetramer)
Riftia pachyptila C1 Hb(24 subunits)
Lumbricus terrestris Erythrocruorin (180 subunits)
Annelida
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HbI
Scapharca inaequivalvis
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Deoxy HbI – 1.6 Å resolution HbI-CO – 1.4 Å resolution
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Cooperative ligand binding in HbI relies on only small subunit rotations
Despite rather localized structural transitions, the R-state is estimated to bind oxygen 300 times more tightly than the T-state.
Unlike binding of oxygen to human hemoglobin crystals, binding of oxygen to HbI crystals is fully cooperative (Mozzarelli et al, 1996).
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Ligation of HbI results in extrusion of Phe 97 from the proximal pocket
Distal His
Distal His
ligand
Proximal His
Phe 97
Phe 97
Proximal His
Mutation of Phe 97 leads to increased oxygen affinity and sharply diminished cooperativity p50
(torr)n
WT 10.0 1.5
F97L 1.0 1.2
F97A 0.3 1.2
F97Y 0.08 1.2
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The isosteric mutation Thr E10 to Val reveals the importance of the observed water cluster for stabilization of the low affinity state
Deoxy HbIp50=10 Torr, n=1.5
Deoxy T72V p50=0.2 Torr, n=1.7
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Raising the osmotic pressure increases the oxygen affinity of HbI, supporting a key role of water molecules in stabilizing the low affinity state of HbI
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Restricting heme movement abolishes allosteric transition
114I
114I
HbI
Mutation of I114F results in low affinity and cooperativity (n=1.05, p50 = 21 Torr).
114F
114F
I114F
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Mutation of distal His to Gln abolishes heme movement and allosteric transition
HbI
E7His
E7His
H69Q
E7Gln
E7Gln
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Key structural transitions with functional ramifications
Heme movement
F4 Phe flipping
Interface waterrearrangment
What is the cascade of structural events?
Are these transitions concerted or sequential?
Do structural kinetic intermediates facilitate
R to T transition?
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CCD
Dye laser
Heat load shutter
2 s chopper
ms shutter
Experimental Setup
e-
BIOCARS 14-IDB, APS
Superbunch ~500ns
Single bunch ~150ps
Cycle time – 3.683 s
X-rays (Undulator)
Time-resolved crystallography to obtain snapshots along the trajectory between high-affinity and low affinity states
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5ns
60s
Subunit A
Fe
CO*
CO
F4F8
F7
E7CD1
CD3
F4
FeF8
F7
CD3
CD1E7
F4
E
E
F
F
CD
CD
F
F
F
F
F4
Fo(light)-Fo(dark) - (Red: -3, Blue: 3)
(Red: -2.5Blue: 2.5)
(Red: -2.5Blue: 2.5)
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Results of difference Fourier refinement, Fo(light) –Fo(dark)
coefficients
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Integrated difference [Fo(light)-Fo(dark)] electron density values
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Change in iron position, based on difference refinementDistance along the parallel and perpendicular components of the heme plane
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Key structural transitions with functional ramifications
Heme movement
F4 Phe flipping
Interface waterrearrangment
What is the cascade of structural events?
Intermediate is formed rapidly (<5ns) upon ligand release, relaxing to T-like structure in microsecond time domain
Are these transitions concerted or sequential?
Key allosteric changes appear to be tightly coupled.
Do structural intermediates facilitate R to T transition?
Rapid disordering of water molecules H-bonded to propionates
appears to lay the foundation for subsequent heme movement.
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Scapharca HbI Deoxy Lamprey HbV
Deoxygenated lamprey hemoglobin oligomerizes in a proton dependent fashion, conferring a strong Bohr effect
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W72’
W72
N79
N79’H73
H73’
E75E75’
E-helix
E’-helix
The primary contacts in the deoxy lamprey HbV dimer involve the E-helices
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Dimerization of Lamprey Hb sterically restricts ligand binding
ProximalHistidine
Distal Histidine
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Lamprey hemoglobin has a very pronounced Bohr effect
From: Antonini et al. (1964) Arch. Biochem. Biophys. 105, 404-408, courtesy of Austen Riggs
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E75’
E75
E31
E31’
R71
R71’
H73
H73’
The strong Bohr effect in lamprey HbV can be accounted for by a cluster of glutamate residues in the interface
Mutations E75Q, Y30H and H73Q support the functional importance of this dimeric structure (Y.Qiu et al. A.F. Riggs (2000) JBC 275, 13517-13528)
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o oo o oo ooHH --
2H+
2O2
o oo o- -
O-O
oo oo- -
O-O
Linkage of proton and oxygen binding in Lamprey Hemoglobin
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Role of Interferon Regulatory Factors (IRFs) in 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
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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.
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IRFs are activated by phosphorylation in the C-terminal domain
P
P P
Cytoplasm
Nucleus
C
N
CBP or p300
P P
DD
P P
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IRF3 IRF5Expression Ubiquitous Complex
Autoinhibition Tight Looser
Stimulated by Viral infection
Viral Infection
p53
Interferon
Stimulates Interferon proinflammatory
cytokines
Tumor suppresors
Interferon
Medical Importance
Antiviral activity Tumor Suppression
Autoimmune disease
Antiviral activity
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Ser/Thr 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 is constitutively expressed in all cell types and acts as a molecular sentry for viral infection.
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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
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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
*
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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
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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
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IRF-5 (222-467) S430D Dimer
Hel
ix 5
Hel
ix 5
N
N
C
C
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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
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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 (222-467) in solution
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Disruption of dimerization by mutation of interface residues inhibits full length IRF-5 activation
HEK293 Cells
IFN
luce
rfer
ase
(F
old
In
du
ctio
n)
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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
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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 (173-427) dimer in solution
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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 IRF-
5 dimer as representing the active state of IRF-3
MockNDV
IFN
luc
erf
era
se
(F
old
In
du
cti
on)
IFN
luc
erf
era
se
(F
old
In
du
cti
on)
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(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)
Phosphorylation
IRF activation, dimerization and CBP binding
C-term
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(Morphing CNS script from the Yale Morph Server, http://molmovdb.org)
Phosphorylation
IRF activation, dimerization and CBP binding
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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
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Hemoglobin projects
James Knapp Holly Heaslet
Animesh Pardanani Michele Bonham
Candace SummerfordMichael Omartian
Jeff NicholsQuentin Gibson
BioCARS – Univ. of ChicagoTime-resolved Crystallography
Vukica SrajerReinhard Pahl
$ - NIH Kai Lin
IRF project
Weijun ChenSuvana Lam
Hema SrinathBrendan Hilbert
Celia Schiffer
Kate FitzgeraldZhaozhao Jiang
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IRF-5Autoinhibition of IRF-5 is less tight than that for the ubiquitously expressed IRF-3.
IRF-5 is activated by:• infection by some viruses• type I interferon• tumor suppressor p53
IRF-5 activates• type I interferon• proinflammatory cytokines, including TNF-, IL-12 and IL-6• 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• resistance to endotoxin shock• susceptibility to tumors
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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
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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
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Helix 5 plays key alternate roles in IRF autoinhibition and dimerization.
IRF-5 dimerIRF-3 monomer
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Sample Diffraction for HbI
Most images result from 30 to 50 flashes allowing CO rebinding between each flash
Crystals and Data Collection
Dithionite +Phosphate
Polyvinyl filmover HbI crystal
Cement
Cement