The Oxygen Sensor
Mechanism of PHD
2020/2/6
Literature Seminar
B4 Fujiyoshi

Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residue of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

1Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residue of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

2The Novel Prize in Physiology or Medicine 2019
Discovery: How cells sense and adapt to oxygen availability

William G. Kaelin Jr
February 6, 2020

Sir Peter J. Ratcliffe(The Nobel Prize in Physiology or Medicine 2019)

Gregg L. Semenza
Kohei Fujiyoshi

3Question: How do cells sense and adapt to the oxygen availability?

O2

FOOD

ATP

Hypoxia?
Increase of erythropoietin in kidney

February 6, 2020

William G. Kaelin Jr., Sir Peter J.
Ratcliffe and Gregg L. Semenza
identified molecular machinery that
regulates the activity of genes inresponse to varying levels ofoxygen.

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

4Overall mechanism of hypoxia response
O2

February 6, 2020

PHD

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

5Physiology and pathology related to hypoxia response

February 6, 2020

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

6

Main topic: prolyl hydroxylase domain (PHD)
O2February 6, 2020

PHD

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

7

Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residue of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

8Transcriptional activation by HIF-α in hypoxia
O2February 6, 2020

PHD

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

9Transcriptional activation by the interaction of HIF1α and p300/CBPHIF1α in Hypoxia
1. No degradation
2. Nucleus translocation
3. Complex formation with HIFβ,
CBP/p300 and other co-factors
4. Transcription activation

February 6, 2020

Chen, C., and Lou, T. (2017).
Oncotarget;Vol 8, No 28.

Kohei Fujiyoshi

10

HIFα target genes for hypoxia response

Angiogenesis
O2 supply
Metabolism
February 6, 2020

Stemness
Self renewal
Redox Homeostasis

Proliferation

EMT

Apoptosis

Metastasis
Invasion

Dengler, V.L., Galbraith, M.D., and Espinosa, J.M.
(2014). Crit. Rev. Biochem. Mol. Biol. 49, 1–15.

Kohei Fujiyoshi

11

HIFα modulate cellur metabolism.
In hypoxia, the aerobic metabolism is switchedto the anaerobic metabolism.

①HIFα inducts
1. GLUT1
1. To increase the glucose transportation(much glucose for anaerobicmetabolism)②③
February 6, 2020

2. PDK
1. To inhibit the conversion from pyruvateto acetylCoA (Stop TCA cycle)
3. LDHA
1. To convert pyruvate to lactate (OxidizeNADH)

Majmundar, A.J., Wong, W.J., and Simon, M.C. (2010). Mol. Cell 40, 294–309.

Kohei Fujiyoshi

12Effect of HIFα on multiple steps of cansar development
Rapidly proliferating cancer cells may outgrow their vascular
network, limiting O2 diffusion within the tumor.
→ Hypoxic stress→ HIFα expression and the downstream activation of thehypoxic stress response are widespread in many cansers.
1. Canser stem cell
2. Proliferation and suvival
3. Metabolism
4. Angiogenesis
5. Cell infiltration
6. Invasion and metastasis
February 6, 2020

Majmundar, A.J, et al.,(2010)

Kohei Fujiyoshi

13

VHL recognition of HIF-α and proteosomal degradation
O2

February 6, 2020

PHD

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

14

HIFα is ubiquitination by E3 ligase and degraded by proteasome.

①②

HIFα in normoxia
1. Hydroxylation of proline
2. Recognition of hydroxyproline by VHL
3. Ubiquitibnation by E2③February 6, 2020④

4. Degradation by proteasome

Buckley, D.L., Van Molle, I., Gareiss, P.C., Tae, H.S.,
Michel, J., Noblin, D.J., Jorgensen, W.L., Ciulli, A., andCrews, C.M. (2012). J. Am. Chem. Soc. 134, 4465–4468.

Kohei Fujiyoshi

15

PHD is an oxygen sensor in hypoxia response
O2February 6, 2020

PHD

(The Nobel Prize in Physiology or Medicine 2019)

Kohei Fujiyoshi

16

Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residue of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

17

Fe(Ⅱ)/2OG dependent oxygenase
Hydroxylation of Pro in procollagen

Hydroxylation of Pro and Gln in HIFαHydroxylation of Lys in histoneOOHOOHO

February 6, 2020

2-oxoglutarate (2OG)

Islam, M.S., Leissing, T.M., Chowdhury, R., Hopkinson, R.J.,
and Schofield, C.J. (2018). Annu. Rev. Biochem. 87, 585–620.
Kohei Fujiyoshi

18

General catalytic cycle for Fe(Ⅱ)/2OG-dependent oxygenase
2OGOO

HO

2-oxoglutarate (2OG):
decarboxylation to succinate

OH
O

Fe(Ⅱ): Fe(Ⅳ)=O a reactive intermediate
Two His and one Asp or Glu: Fe(Ⅱ) ligand
Water: Fe(Ⅱ) ligand replaced to O2

February 6, 2020

Flashman, E., Hoffart, L.M., Hamed, R.B., Bollinger Jr, J.M.,
Krebs, C., and Schofield, C.J. (2010). FEBS J. 277, 4089–4099.

Kohei Fujiyoshi

19Essentials for O2 dependent reactivity in PHD
Oxygen sensor

O2dependent
reactivity

2OG & Substratedependency

Binding substrate&Release substrate

Slow
O2-H2O
replacement

Stable2OG/Fe(Ⅱ)
complexkcat ⋅[E]T ⋅[O2 ] kcat ⋅[E]T ⋅[O2 ]v ([S],[2OG],[O2 ]) =!
K m ≫ [O2 ])(K m + [O2 ]Km
February 6, 2020

Kohei Fujiyoshi

20

The rate-determining steps for O2 dependent reactivityBinding
substrate

Releasesubstrate

February 6, 2020

Stable2OG/Fe(Ⅱ)
complexSlow
O2-H2O
exchange

Oxygen sensor

O2dependent
reactivity

2OG & Substratedependency

Binding substrate&Release of product

Slow
O2-H2O
exchange

Stable2OG/Fe(Ⅱ)
complexFlashman, E., et al., (2010).

Kohei Fujiyoshi

21

Stable binding of substrate andeasy release of product

Substrate binding

Binding of substrate to enzyme generally stimulates areaction in other hydroxylase. (TauD; w/ : w/o = 1000 : 1)
● 2OG to succinate with CODD
kcat = 0.018 s–1○ 2OG to succinate without CODD
kcat = 0.0006 s–1 → 30-fold slower(● CODD hydroxylation: kcat = 0.013 s–1)
PHD:Fe(Ⅱ):2OG(CODD:C-terminal oxygen-dependent degradation domain)→ In PHD2, substrate binding may
not be a rate-determiniting step.

February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

22

Stable 2OG/Fe(Ⅱ) complex

Previous EPR analysis showed that unstable 2OG
chelation caused uncouple 2OG decarboxylaiton andproduced reactive Fe ion, which was quenched by
reductant such as ascorbate.
2OG decarboxylation is coupled to production ofsuccinate and CODD hydroxylation.
→ 2OG/Fe(Ⅱ) complex is quite stable.
2OG binding may not be a rate-determining step inPHD2February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

23

O2 dependent reactivity= PHD1= PHD2= PHD3

Km for O2
PHD2: 250 µM
Other oxygenase: 40 µM
PHD2 has a higher Km for O2 than other oxygenases.
→ O2 binding may be a rate-determining step in PHD2.

February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

24

The rate-determining steps for O2 dependent reactivity
Binding of
substrate

Release ofsubstrate

Stable2OG/Fe(Ⅱ)
complex
Slow O2-H2O
exchange

Oxygen sensor

O2 dependent
reactivity

2OG dependency
Substrate dependency

Stable binding of
substrate and easy
release of productSlow O2-H2O
exchange

Stable 2OG/Fe(Ⅱ)
complex

What structural properties
do contribute to the O2
dependent reactivity?
February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

25

Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residue of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

26

The structure of PHD2.NOG.Mn(Ⅱ).CODDOOOHOOH

HNHOO

O

2OGNOGOOH

NOG (Orange) and Mn(Ⅱ) (Purple) are substituited for
2OG and Fe(Ⅱ) as non-reactive analogs.
CODD (HIF1α peptide including Pro-564, Yellow) isused as a substrate.
The tPHD fold comprises four α helices and ten βstrands of which eight form a double-stranded β helix(DSBH, dark blue).
Chowdhury, R., McDonough, M.A., Mecinović, J.,Loenarz, C., Flashman, E., Hewitson, K.S., Domene, C.,
and Schofield, C.J. (2009). Structure 17, 981–989.
February 6, 2020Three of four α helices (α1, α2, and α3) pack along themajor β sheet and stabilize the DSBH.
Kohei Fujiyoshi27

β2β3/loop anchored by a salt bridge

β2β3/loop
The β2β3/loop of PHD envelops the CODD
substrate and stabilizes the substrate-enzyme
complex.
→ HIFα stably binds PHD.
Salt bridgeΒ2β3/loop semmed to be anchored by a salt
bridge of R252 and D254.

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

28

The β2β3/loop is important for substrate binding.
PHD2.CODD (Green)
・R252 and D254 form a salt bridge.
PHD2.Fe(Ⅱ).inhibitor (Cyan)
・The inhibitor(salmon) seemed toprevent the salt bridge formation.
PHD2 R252 and D254 variants
couldn’t hydroxylate CODD Pro.
→ The β2β3/loop stabilized by thesalt bridge of D254 and R252
contributes much to substrate binding

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

29The conformational change of Pro564 by hydroxylation

Pro-564CODD : C4 endo → Hyp-564CODD : C4 exo conformtion.
Endo to Exo switch might contribute to the release of the product from PHD active site.
VHL seemed to recognize exo conformation of Hyp-564CODD but cannot recognize endo
conformation of Pro-564CODD.
February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

30Summary of structural analysis of PHD2β2β3/loop

Salt bridge

The β2β3/loop anchored by a salt bridge contribute much to the stable substrate binding.
Endo to exo conformational switch is important for release of product and recognition by VHL-E3ligase.
February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

31Summary of structural analysis of PHD2The comformation of the β2β3/loop of PHD2 isimportant to enclose the Pro-564 region ofHIFα.
The salt bridge comprised of R252 and D254
stabilizes the β2β3/loop conformation.
The Pro-564 ring conformation switches endo
to exo by hydroxyation.

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

32

Contents• Introduction• The Novel Prize in Physiology or Medicine 2019
• Mechanism of hypoxia response• Basic characteristics of PHD• Structural analysis of PHD2
• Contribution of residues of PHD2 to O2 dependent reactivity• SummaryFebruary 6, 2020

Kohei Fujiyoshi

33

2OG is stabilized by R383

Salt bridge between R383 and 2OG
stabilize the 2OG/Fe(Ⅱ) complex
The stability of 2OG/Fe(Ⅱ) complex
inhibit a rearrangement of 2OG andwater to bring water close to Pro.

February 6, 2020

Tarhonskaya, H., Chowdhury, R., Leung, I.K.H., Loik, N.D.,
McCullagh, J.S.O., Claridge, T.D.W., Schofield, C.J., andFlashman, E. (2014). Biochem. J. 463, 363–372.

Kohei Fujiyoshi

34

The metal-bound water is stabilized by Asp-315.

Hydrogen bond between D315 andmetal ligated water stabilize the watermetal coordination, which inhibits
O2-H2O exchange.

February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

35

Question Ⅱ

How do these residue
contribute to the O2
dependent reactivity?

February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

36

CODD hydroxylation of PHD2 variants

Y303F and Y329F (2OG):
H313D, D315H, H374E and H374R (Fe(Ⅱ)):
R383K (2OG):
D315E (Fe(Ⅱ)):
February 6, 2020

Tarhonskaya, H,. et al.(2014)

No significant change
Loss of catalytic activity
Moderately decrease
Moderately increase
Kohei Fujiyoshi

37Binding Fe(Ⅱ) and 2OG to the PHD2 variants
PHD+2OG+Fe(Ⅱ)
PHD+2OG
PHD

R383K: Reduced affinity for 2OG and no change in the affinity for Fe(Ⅱ)
Kd for 2OG with CODD is lower than without CODD.
D315E: The stability of PHD2.Fe(Ⅱ) complex was decreased.
February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

38

The Km for 2OG and CODD and kcat values of PHD2 variants

R383K Km(2OG): ~3-fold higher, Km(CODD): ~2-fold higher, kcat: lower
R383 contribute much to the stability of 2OG/Fe(Ⅱ) complex and some to substrate dependent
reactivity.
D315E Km(2OG): slightly higher, Km(CODD): No change, kcat: slightly higher
D315 does not contribute to both of them.
February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

39

Accumulation of Fe intermediate rearranged in chelation

R383K: faster than WT with CODD
No rearrangement without CODD
→ R383 contributes to the stability of the 2OG/Fe(Ⅱ) complex

February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

40

O2-dependency of PHD2 WT and variants

R383K Km(O2): Couldn’t be determined (estimated >450 µM, WT estimated >450 µM)
D315E Km(O2): 200 30 µM lower than WT and R383K→ D315 contributes much to the high Km for O2

February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

41

CODD hydroxylation and uncoupled 2OG decarboxylationCODD hydroxylation

R383K: CODD hydroxylation and production of succinate is similar to WT→ The 2OG/Fe(Ⅱ) complex in R383K without is as stable as WT.
D315E: CODD hydroxylation is faster than WT and production of succinate without CODD
increase.
→ The uncouple reaction proceed, which means the stability of 2OG/Fe(Ⅱ) complex is lost.
February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

42Summary of kinetic analysis of PHD2 variants R383K

Tarhonskaya, H,. et al.(2014)

Compare to WT

R383K

D315E

CODD hydroxylation

DecreaseIncrease

Binding Fe(Ⅱ)

Same

Decrease (flexibility increase)

Binding 2OG

Decrease

Km (2OG)

Decrese(higher w/ than w/o CODD)~3-fold higher

Km (CODD)

No major change

No major change

Km (O2)–LowerFe(Ⅳ)=O accumulation

Faster–Oxidation of 2OG without CODD

Little increase

Much increase

Slightly higher

R383 contributes to stabilize 2OG at the active site.
The rearrange of chelation in R383K is faster than for WT, but no changes in the kinetics ofproduct accumulation (hydroxylated CODD and succinate) was observed.
→ The rearrangement of the 2OG-binding mode may not be a rate-determining step in PHD2catalysed hydroxylation. However, R383 contributes much to the stability of 2OG/Fe(Ⅱ)
complex.
February 6, 2020

Kohei Fujiyoshi

43Summary of kinetic analysis of PHD2 variants D315E

Tarhonskaya, H,. et al.(2014)

Compare to WT

R383K

D315E

CODD hydroxylation

Decrease

Increase

Binding Fe(Ⅱ)

Same

Decrease (flexibility increase)

Binding 2OG

Decrease

Km (2OG)

Decrese(higher w/ than w/o CODD)~3-fold higher

Km (CODD)

No major change

No major changeKm (O2)–Lower

Fe(Ⅳ)=O accumulation

Faster–Oxidation of 2OG without CODD

Little increase

Much increase

Slightly higher

D315 stabilizes the Fe(Ⅱ) at the active site and the iron-ligated water by a hydrogen bond.
CODD hydroxylation by D315E is faster than WT.
Oxidation of 2OG uncoupled to CODD hydroxylation by D315E was observed unlike WT.
→ The bond of the metal-co-ordinated water to metal is weakened, facilitating water releaseand accelarating O2 activation.
This is an important factor in the slow O2 activation of PHD2 catalysis.
February 6, 2020

Kohei Fujiyoshi

44Summary
Oxygen sensor

Binding substrate&Release of productO2dependent
reactivity

SlowO2-H2O
exchange

β2β3/loop

2OG & Substratedependency

Stable2OG/Fe(Ⅱ)
complexSaltbridge

Chowdhury, R., et al., (2009)

Stabilization ofsubstrate by
β2β3/loop

Endo to exo switch
by hydroxylation

Hydrogen bond of
metal coordinatedwater with D315

Salt bridge formedby 2OG and R383

kcat ⋅[E]T ⋅[O2 ]v ([S],[2OG],[O2 ]) !
K m ≫ [O2 ])(Km
Tarhonskaya, H,. et al.(2014)

February 6, 2020

Kohei Fujiyoshi

45Appendix

February 6, 2020

Kohei Fujiyoshi
46

Structure of an HIFα and pVHL complex

Min, J.H., Yang, H., Ivan, M., Gertler, F., Kaelin, W.G., and Pavietich,
N.P. (2002). Structure of an HIF-1α-pVHL complex: Hydroxyproline
recognition in signaling. Science (80-. ). 296, 1886–1889.
February 6, 2020

Kohei Fujiyoshi

47

Crystallization of tPHD2.HIFα Complex
A and B:tPHD2.Fe(Ⅱ).inhibitor.CODDHyp564 complex(tPHD.Fe(Ⅱ).A/B)• It crystallizes in a homotrimeric form.
• The electron density was not observed forhydroxylated Pro-564.
• The reason is that the C-terminal helix (α4)
for a symmetry molecule blocks the activesite.

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

48

Crystallization of tPHD2.HIFα Complex
C and D:
R398A tPHD2.Mn(Ⅱ).NOG.HIF-1αCODD556-574
complex (tPHD2.CODD)• R398A variant destabilize the homotrimer
formation.
• NOG and Mn(Ⅱ) were substituted for 2OGand Fe(Ⅱ), respectively.
• The apparent movement of residues 237254 (β2β3/loop) is proposed to enclose thesubstrate.
ON-oxalylglysine (NOG)

HNHOO
OHO
February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

49

Salt bridge environment

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

50

PHD variants forming salt bridge R252 and D254

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

51

PHD and FIH

February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

52

LXXLAP motif

LXXLAP motif of CODD is positioned inhydrophobic site of PHDFebruary 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

53

Binding Fe(Ⅱ) and 2OG to the PHD2 variants
D315E: The stability ofPHD2.Fe(Ⅱ) complex
decreased.
→ It is inconsistant with thehigh rate of hydroxylation.
Structural analysis of D315E
PHD2.Mn(Ⅱ).Inhibitor complex,
・The flexibility of β2β3
increased
・Two conformation of Glu315
side chain were observed
・The bond of metal-water isweaker.
February 6, 2020

Chowdhury, R., et al., (2009)

Kohei Fujiyoshi

54

2OG turnover and hydroxylation

February 6, 2020

Tarhonskaya, H,. et al.(2014)

Kohei Fujiyoshi

55

Catalyst of Fe(Ⅱ)/2OG-dependent oxygenase2-oxoglutarate (2OG)
Oxidative decarboxylation of 2OG produces
CO2, succinate and Fe(Ⅳ)=O.
OO

HOFe(Ⅱ)OHO

Fe(Ⅳ)=O, a reactive intermediate, cleave thesubstrate C–H bond by hydrogen abstraction
to produce Fe(Ⅲ)–OH and C･ radical.
The C･ radical reacts with a hydroxyl radical
derived from Fe(Ⅲ)–OH and produces C–OHbond.
February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

56

The coordination of Fe(Ⅱ) centerThe Fe(Ⅱ) center of 2OG-dependentoxygenases is normally coordinated by threeprotein derived ligands two His and one Glu orAsp.
2OG binds to the Fe(Ⅱ) in a bidentate manner.
The exchange from coordinated water tooxygen occurs when substrate binds toadjacent to the Fe(Ⅱ) and weaken binding of
the water.

February 6, 2020

Flashman, E., et al., (2010).

Kohei Fujiyoshi

57