Regulation of biological activity through protein dimerization: Crystallographic analyses of primitive hemoglobins and interferon regulatory factors Bill Royer University 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 Chordata Echinodermata Echiura Mollusca Protostomes 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 resolutionHbI-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 Proximal His Mutation of Phe 97 leads to increased oxygen affinity and sharply diminished cooperativity p50 (torr) n WT F97L F97A F97Y

The isosteric mutation Thr E10 to Val reveals the importance of the observed water cluster for stabilization of the low affinity state Deoxy HbI p50=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 HbI Mutation of I114F results in low affinity and cooperativity (n=1.05, p50 = 21 Torr). 114F I114F

Mutation of distal His to Gln abolishes heme movement and allosteric transition HbI E7His H69Q E7Gln

Key structural transitions with functional ramifications Heme movement F4 Phe flipping Interface water rearrangment 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-e- BIOCARS 14-IDB, APS Superbunch ~500ns Single bunch ~150ps Cycle time –  s X-rays (Undulator) Time-resolved crystallography to obtain snapshots along the trajectory between high-affinity and low affinity states

5ns 60  s Subunit A Fe CO* CO F4 F8 F7 E7CD1 CD3 F4 Fe F8 F7 CD3 CD1 E7 F4 E E FF CD F F F F F4 F o (light)-F o (dark) - (Red: -3 , Blue: 3  ) (Red: -2.5  Blue: 2.5  ) (Red: -2.5  Blue: 2.5  )

Results of difference Fourier refinement, F o (light) –F o (dark) coefficients

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

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

Key structural transitions with functional ramifications Heme movement F4 Phe flipping Interface water rearrangment 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 HbIDeoxy Lamprey HbV Deoxygenated lamprey hemoglobin oligomerizes in a proton dependent fashion, conferring a strong Bohr effect

W72’ W72 N79 N79’ H73 H73’ E75 E75’ 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 Proximal Histidine Distal Histidine

Lamprey hemoglobin has a very pronounced Bohr effect From: Antonini et al. (1964) Arch. Biochem. Biophys. 105, , 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, )

o o o o o o o o H H - - 2H + 2O 2 o o o o -- O-OO-O o o o o -- O-OO-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 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 or p300 PP DD PP

IRF3IRF5 ExpressionUbiquitousComplex AutoinhibitionTightLooser Stimulated byViral infection Viral Infection p53 Interferon  Stimulates Interferon  proinflammatory cytokines Tumor suppresors Interferon  Medical Importance Antiviral activityTumor Suppression Autoimmune disease Antiviral activity

Ser/Thr 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 is constitutively expressed in all cell types and acts as a molecular sentry for viral infection.

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

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 *

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 ( ) S430D Dimer Helix 5 N N C C

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 full length 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 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 Mock NDV IFN  lucerferase (Fold Induction )

(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

Hemoglobin projects James Knapp Holly Heaslet Animesh Pardanani Michele Bonham Candace Summerford Michael Omartian Jeff Nichols Quentin Gibson BioCARS – Univ. of Chicago Time-resolved Crystallography Vukica Srajer Reinhard Pahl $ - NIH Kai Lin IRF project Weijun Chen Suvana Lam Hema Srinath Brendan Hilbert Celia Schiffer Kate Fitzgerald Zhaozhao Jiang

IRF-5 Autoinhibition 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 ( ) 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

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

Helix 5 plays key alternate roles in IRF autoinhibition and dimerization. IRF-5 dimer IRF-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 film over HbI crystal Cement