Heme-sensors Associated with Circadian

Rhythms and Transcription

1

Sensor domain Functional domain

Protein structural

changes

Sensor domain

Heme binding

Functional domain Catalysis, DNA

binding

Heme-responsive heme-sensor proteins

N N

N N

O

O

O

O

Heme as a

signal

Fe

2

Heme association/dissociation regulates functions.

HRI: Ser/Thr kinase/Protein synthesis -S-heme

NPAS2: Transcription regulation/Body clock -S-heme

mPer2: “ -S-heme

Bach1: Transcription regulation/Heme metabolism -S-heme

IRP2 : Protein degradation -S-heme

Irr: Protein degradation -S-heme

E75 or Rev-erba: Pheromone receptor /Metabolism -S-heme?

DGCR8: RNA binding/Degradation -S-heme

Slo1 BK channels: Calcium channel -S-heme

The N-end role pathway: arginyl-transferase -S-heme?

TrpRS: -N-heme 3

Circadian rhythms (Body clock)

Sleep

Feed Awake

Photo:http://www.ngrl.co.jp/koutarou

4

5

E-box

BMAL1 NPAS2

per, cry …

Forebrain

Mouse

brain

Oscillation Mechanism

BMAL1 NPAS2

E-box

heme CO

E-box

heme

HO heme

E. M. Dioum et al., Science 298, 2385 (2002)

Transcriptional Control

SCN

E-box per, cry

Negative factor

BMAL1 CLOCK

Forebrain

E-box per, cry

BMAL1 NPAS2

BMAL1-NPAS2-DNA(E-box) Interaction

6

E box

NPAS2

Full length

Heme

CO

Heme

NADPH E box

bHLH PAS-A PAS-B

Heme Heme

1 78 240 416 816

BMAL1 Full length bHLH PAS-B PAS-A

1 67 140 334 447 626

5’-GGGGCGCCACGTGAGAGG-3’

3’-CCCCGCGGTGCACTCTCC-5’

E box

Transcription

NPAS2: neuronal PAS domain protein 2

McKnight, S. et al. Science 298, 2385 (2002). 7

bHLH/PAS-A and PAS-A domains of NPAS2

bHLH/PAS-A bHLH PAS-A

1 78 240 416 816

bHLH PAS-A PAS-B Full

length

PAS-A PAS-A Present study

bHLHPASA

8

Uchida, T. et al. J. Biol. Chem. 280, 21358 (2005).

Resonance Raman spectra of WT and C170A species of the

ferric NPAS2 PAS-A domain in the low frequency region

9

Uchida, T. et al. J. Biol. Chem. 280, 21358 (2005).

Hypothetical model of the heme coordination structure

of the PAS-A domain

Fe(III)-Cys

Fe(II)-His

10

11

DNA Binding Analysis of the bHLH/ PAS-

A Domain of BMAL1 and NPAS2

E-box

BMAL1 BMAL1

E-box

NPAS2 NPAS2

E-box

BMAL1 NPAS2

A B

Heme Heme NPAS2

bHLH dimerization DNA binding ▼

PAS secondary dimerization ▼

BMAL1

Objectives: Investigations of interactions between E-box and proteins

associated with circadian rhythms using the QCM method.

B A

? ?

bHLH/PAS-A and PAS-A domains of NPAS2

bHLH/PAS-A bHLH PAS-A

1 78 240 416 816

bHLH PAS-A PAS-B Full

length

PAS-A PAS-A

bHLHPASA

Present study

12

Affinix Q

Sensor chip

Sample injection

Personal Computer

Oscillation Circuit

Frequency Counter

A DNA duplex-immobilized 27 MHz QCM

Okahata et al. 1998

Quartz-crystal Microbalance (QCM)

Interactions between DNA and NPAS2

13

Mechanism of QCM

Oscillation frequency

Frequency counter

Computer calculation

5’ GGGGCGCCACGTGAGAGG3’

3’ CCCCGCGGTGCACTCTCC 5’

E-box

NeutroAvidine

Biotin

Au

CONH

OPHOOO

1400

1200

1000

800

600

400

200

0

DF

Hz

30002500200015001000

Time (s)

biotin labeled Ebox2

Ebox1Ebox1

Ebox1

ΔF (Hz) vs Time (s)

O

OPO

O

14

⇒ Holo-NPAS2 binds to the E-box in the absence

of BMAL1.

Mukaiyama, Y. et al. FEBS J. 273, 2528 (2006).

Binding of holo-NPAS2 to the E-box

AU

Quartz

Heme

Interactions between DNA and NPAS2

-3000

-2000

-1000

0

1000

D

F (

Hz)

12000 10000 8000 6000 4000 2000

time (sec)

holo-NPAS2

holo-NPAS2

holo-NPAS2

holo-NPAS2

holo-NPAS2

BSA

15

⇒ Apo-NPAS2 does not bind to the E-box in the

absence of BMAL1.

Binding of apo-NPAS2 to the E-box

AU Quartz

NPAS2

1500

1000

500

0

-500

D F

( H

z)

4000 3500 3000 2500 2000 1500

time ( sec)

apo-NPAS2

BSA apo-NPAS2 apo-NPAS2 apo-NPAS2

apo-NPAS2

16

No binding of holo-NPAS2 to the mutated sequence

⇒ Holo-NPAS2 does not bind to DNA with the

mutated sequence.

2500

2000

1500

1000

500

0

-500

-1000

D

F, H

z

4000 3500 3000 2500 2000 1500

time, sec

holo-NPAS2

BSA

holo-NPAS2 holo-NPAS2

AU

Quartz

3’ CTGCAG5’

5’ GACGTC3’

mutated sequence

Heme

17

5’ GGGGCGCCACGTGAGAGG3’

3’ CCCCGCGGTGCACTCTCC 5’

The PAS-A domain does not bind to the E-box.

E-box

PAS-A domain of NPAS2

-1000

-500

0

500

1000

D F

(H

z)

2500 2000 1500 1000 500 0

Time (sec)

PAS-A

PAS-A

PAS-A

PAS-A PAS-A

18

Heme

NADPH

Heme

bHLH PAS-A

The bHLH domain interacts with the PAS domain.

E box DNA

E box DNA

NADPH facilitates DNA binding? Heme facilitates

DNA binding.

19

Heme dissociation from the bHLH/PAS-A domain of NPAS2

Protein kon(M-1s-1) koff(s-1) Kd(M)

bHLH/PAS-A 3.3 x 107 5.3 x 10-3 1.6 x 10-10

p22HBP 1.0 x 108 4.4 x 10-3 4.4 x 10-11

HRI 1.1 x 107 1.5 x 10-3 1.4 x 10-10

SWMb 7.0 x 107 8.4 x 10-7 1.2 x 10-14

* Kd = koff/kon

0.27

0.26

0.25

0.24

0.23

0.22

Ab

sorb

an

ce a

t 4

10

nm

40003000200010000

Time (s)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

Ab

sorb

an

ce

460440420400380

Wavelength (nm)

Mb: 30 μM

bHLH/PAS-A: 3 μM

20

E box DNA NADPH

E box DNA NADPH

BMAL1

?

Fe(III)-S-Cys: Both PAS-A and bHLH-PAS-A domains.

Fe(II)-N-His: Redox-dependent ligand switching.

Fast dissociation of the Fe(III) complex from bHLH-PAS-A.

Heme-bound NPAS2 alone binds to DNA without BMAL1.

Does NPAS2 compete with NPAS2/BMAL1 in DNA binding and regulate transcription? Mukaiyama, Y. et al. FEBS J. 273, 2528 (2006).

21

218-416 + -

218-346 + +

160-346 ++ - 160 346

218 416

218 346

Expression, Yield

241-416 ++ ++

241 416

bHLH PAS-A PAS-B

pET28a

Nde I Sal I

Expression

Construction of NPAS2

Characterization of the Isolated NPAS2 PAS-B Domain

Mouse NPAS2

PAS-B binds heme.

Fe3+: 6-coordinated LS

His335 + unknown ligand

22 22

23

The ligand trans to CO is

histidine.

Fig. 2-10 Correlations between νFe-C vs νC-O

Inverse Correlation between the νFe-C versus νC-O

HN

N

Fe

540

520

500

480

460

νF

e-C

(cm

-1)

200019801960194019201900νC-O (cm

-1)

Mt Hb

Sw Mb pH 7.0 Bj FixL

CooA Ec DOS PAS

PAS-B

Fe-S

Fe-N

His335

Koudo et al. FEBS J. 272, 4153 (2005).

23

Reciprocal Regulation of Circadian Clock

and Heme Biosynthesis

Kaasik et al. Nature 2004

PER2 NPAS2

BMAL1

PER2

Per2

PER2 Fe

NPAS2

NPAS2

PER2

Alas1

ALAS1

Fe

HO Fe

CO

BMAL1

BMAL1

Fe

24

Heme

E-box

NPAS2 Per2

Forebrain

BMAL1

Kaasik et al., Nature 430, 467 (2004)

Transcription

Translation

?

25

mouse Per2

Full length

1

Heme

Heme

PAS-A

?

?

1257 327 509

PAS-B

PAS-A

1 327 mouse Per2

PAS-A Present study

Kitanishi, K. et al. Biochemistry 47, 6157 (2008).

26

27

1 . 0

0 . 5

0 . 0

Ab

so

rb

an

ce

a

t

42

6

nm

2 . 01 . 51 . 00 . 50 . 0

F e ( I I ) h e m e / a p o m P e r 2 - P A S - A

PAS-A : Fe(III) hemin = 1:1

0 . 5

0 . 4

0 . 3

0 . 2

0 . 1Ab

so

rb

an

ce

a

t

42

0

nm

2 . 01 . 51 . 00 . 50 . 0

F e ( I I I ) h e m i n / a p o m P e r 2 - P A S - A

Heme Binding to mPer2-PAS-A Fe(III) hemin titration (difference spectra)

Fe(II) heme titration

0 . 6

0 . 4

0 . 2

0 . 0

DA

bs

or

ba

nc

e

7 0 06 0 05 0 04 0 03 0 0

W a v e l e n g t h ( n m )

4 2 0

1 . 5

1 . 0

0 . 5

0 . 0

Ab

so

rb

an

ce

7 0 06 0 05 0 04 0 03 0 0

W a v e l e n g t h ( n m )

4 2 6

28

Optical Absorption Spectra

Soret (nm) Visible (nm)

Fe(III) 365, 421 536

Fe(II) 425 529, 558

Fe(II)-CO 420 538, 565

Fe(III) Fe(II) Fe(II)

CO

Fe(III), Fe(II) and Fe(II)-CO complexes : 6cLs

1 . 0

0 . 8

0 . 6

0 . 4

0 . 2

0 . 0

Ab

so

rb

an

ce

7 0 06 0 05 0 04 0 03 0 0W a v e l e n g t h ( n m )

x 5

F e ( I I I ) F e ( I I ) F e ( I I ) - C O

29

0 . 6

0 . 5

0 . 4

Rh

om

bi

ci

ty

|

R

/

µ

|

6543T e t r a g o n a l i t y | µ / |

H i s / H i s

H i s / C y sC y s / O H -

EPR Spectrum

Axial ligands of mPer2-Fe(III)-PAS-A are

Cys and OH-(?).

mPer2-PAS-A NPAS2-bHLH-PAS-A

4 0 03 0 02 0 01 0 0

M a g n e t i c F i e l d ( m T )

2 . 4 42 . 2 71 . 9 04 . 2 86 . 0 9*

Fe(III)

S-

Cys

OH- ?

30

Hg2+ Titration

Fe(III)

S-

Cys

Hg2+

Fe(III)

S-

Cys

Hg2+

Thiolate is the potential axial ligand for mPer2-Fe(III)-PAS-A.

Fe(III)

S-

2-mercaptoethanol

N ?

0 . 8

0 . 7

0 . 6

0 . 5

0 . 4A

bs

or

ba

nc

e

4 4 04 2 04 0 03 8 03 6 03 4 0W a v e l e n g t h ( n m )

F e ( I I I ) + H g 2 + + E D T A

+ 2 - M e

4 2 14 2 1

4 1 2

4 2 1

421 nm 421 nm 412 nm

31

Fe(III) Dissociation from mPer2-PAS-A

Fe(III) Fe(III)

Holo mPer2-PAS-A

(421 nm)

+

Holo myoglobin

(412 nm)

+

Apo mPer2-PAS-A

koff (s-1)

mPer2-PAS-A 9.7 x 10-4

NPAS2-bHLH-PAS-A 5.3 x 10-3

HRI 1.5 x 10-3

SW Mb 8.4 x 10-7

Apo myoglobin

(H64Y/V68F)

Fast Fe(III) dissociation

0 . 8

0 . 6

0 . 4

0 . 2

Ab

so

rb

an

ce

4 6 04 4 04 2 04 0 03 8 03 6 0

W a v e l e n g t h ( n m )

4 1 2

0 . 7 8

0 . 7 6

0 . 7 4

0 . 7 2

A4

12

4 0 0 03 0 0 02 0 0 01 0 0 00

T i m e ( s )

32 Fe(III) transferred from NPAS2 to mPer2. 〜10 min-1

Fe(III) Transfer from NPAS2 to mPer2

NPAS2

Fe(III)-hemin

mPer2

4 M heme-bound NPAS2 + 4, 8, 12, 16 and 20 M apo mPer2

0 . 2

0 . 1

0 . 0

Ab

so

rb

an

ce

7 0 06 0 05 0 04 0 03 0 0

W a v e l e n g t h ( n m )

4 1 9

4 2 2 N P A S 2 N P A S 2 + m P e r 2

33

mPer2

Fe(III)-hemin

NPAS2

No Fe(III) Transfer from mPer2 to NPAS2

Fe(III) does not transfer from mPer2 to NPAS2.

4 M heme-bound mPer2 + 4, 8, 12, 16 and 20 M apo NPAS2

1 . 0

0 . 8

0 . 6

0 . 4

0 . 2

0 . 0

Ab

so

rb

an

ce

7 0 06 0 05 0 04 0 03 0 0

W a v e l e n g t h ( n m )

4 2 1

m P e r 2

m P e r 2 + N P A S 2

Heme

E-box

NPAS2 mPer2

Forebrain

BMAL1

Transcription

Translation

Mechanism of Circadian Rhythms

Heme

Hypothetical Heme Coordination Structure of mPer2-PAS-A

S- Cys 215

Fe(III)

OH- ?

Kitanishi, K. et al. Biochemistry 47, 6157 (2008). Editor-selected Papers in Cell Biology from Biochemistry 17/1800

34

35

Cys His Cys

JBC 280, 21358 (2005), FEBS J. 272, 4153 (2005), ibid., 273, 2528 (2006)

Biochemistry 477, 6157 (2008), BBA 1814, 326 (2011), J. Inorg. Biochem.

108, 188 (2012), Chapters Circadian Rhythms: Biology (Nova Science) (2012)

36

A proposed model for the role of heme binding in hPer2/hCry1 complex

formation is depicted. Ub: ubiquitin; Fe3+: ferric heme: Fe2+: ferrous heme.

Carla Finkielstein et al. Mol. Cell Biol. 28, 4697 (2008)

A Novel Heme Regulatory Motif Mediates Heme-dependent

Degradation of the Circadian Factor Period 2

HRI, NPAS2, mPer2, mPer1? : Heme-sensor proteins?

PAS-A-Fe(III) heme-S-Cys : Thiolate coordination

Fast Fe(III) heme dissociation

PAS-A-Fe(II) heme-N-His: Redox dependent ligand

switching

37

38

Cry Per2 Heme Per2 Heme

NPAS2 BMAL1

E-box Per, Cry

Heme

Transcription

Translation

Nucleus

Cytoplasm

Cry

mRNA DNA

Protein Post-translational

modification

Transcriptional

repression

2 Kitanishi, K. et al. Fig. 2

39

Per2

E-box

BMAL1 NPAS2

per, cry …

Heme

Heme

heme transfer

Per2

P P

degradation

40

Molecular Mechanism of Ocsillation System of mammalian

mPER3 mPER1

mPER3

mPER1

mPER2 mCRY2

mCRY1

mTIM mPER3

mPER2 mPER3

mPER3

mPER2

mPER1

degradation

CKⅠε

Phospholyration

E box

BMAL1 CLOCK Clock Oscillation genes

mPer1

mPer2

mPer3

mTim

mCry1

mCry2

CKⅠε

degradation nucleus cytoplasm

Molecular biology of heme transfer must be done.

41