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

Sperm whale myoglobin(Structure by John Kendrew, illustration by Irving Geis)

Molluscan hemoglobin subunit

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

HbI

Scapharca inaequivalvis

Deoxy HbI – 1.6 Å resolution HbI-CO – 1.4 Å resolution

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).

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

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

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

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

Mutation of distal His to Gln abolishes heme movement and allosteric transition

HbI

E7His

E7His

H69Q

E7Gln

E7Gln

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?

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

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)

Results of difference Fourier refinement, Fo(light) –Fo(dark)

coefficients

Integrated difference [Fo(light)-Fo(dark)] electron density values

Change in iron position, based on difference refinementDistance along the parallel and perpendicular components of the heme plane

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.

Scapharca HbI Deoxy Lamprey HbV

Deoxygenated lamprey hemoglobin oligomerizes in a proton dependent fashion, conferring a strong Bohr effect

W72’

W72

N79

N79’H73

H73’

E75E75’

E-helix

E’-helix

The primary contacts in the deoxy lamprey HbV dimer involve the E-helices

Dimerization of Lamprey Hb sterically restricts ligand binding

ProximalHistidine

Distal Histidine

Lamprey hemoglobin has a very pronounced Bohr effect

From: Antonini et al. (1964) Arch. Biochem. Biophys. 105, 404-408, courtesy of Austen Riggs

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)

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

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

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 or p300

P P

DD

P P

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

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.

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

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

*

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 (222-467) S430D Dimer

Hel

ix 5

Hel

ix 5

N

N

C

C

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 (222-467) in solution

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)

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 (173-427) dimer in solution

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)

(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

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

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

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

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

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

IRF-5 dimerIRF-3 monomer

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