1

Ec DOS: Escherichia coli Direct Oxygen Sensor

2

Sensor domain

(Heme)

Functional domain

Protein

structural

change

Sensor domain

(Heme)

Functional domain Regulation of

catalysis and

transcription

Signal

(O2, NO, CO etc.)

or redox state

Heme-based gas sensor protein

FixL, HemAT, sGC

CooA, Ec DOS

3

FixL O2 ass/disso His kinase/nitrogen fixation

Ec DOS Heme redox, O2 Phosphodiesterase

HemAT O2 ass/disso Methylation/aerotaxis regulation

sGC NO ass/disso Guanylate cyclase/smooth muscle viagla

CooA CO ass/disso Transcription regulation

NPAS2 CO ass/disso Transcription regulation

CBS CO ass/disso Cystathionine -synthase

Heme-based gas sensor proteins

Fig. 1. Families of heme-based sensors. A distinctive heme-binding domain defines each

family of sensors. Subgroups (red boxes) within the families couple their heme-binding

domain to different transmitters for signal transduction. Those proteins specifically named are

ones that have been purified and established as heme proteins. The physiological functions, if

known, are highlighted in green. The last line in each category notes the numbers and

kingdom of additional members expected from sequence homology.

O2 Sensor

O2 Sensor

CO Sensor

CO Sensor

O2 Sensor

NO Sensor

Fig. 2. Classification schema of biological heme-based sensors. Heme-based sensors and their

domain organization are illustrated. Individual globin-coupled sensors are assigned to their

respective class based on the known/putative functions of their signaling domains. The name

ERERQR is a name given to the domain between the globin and DUF1 domain and based on

the ERERQR motif it contains[7]. Color-coding corresponds to the SMART domains as

represented in the figure key.

6

Heme-bound domain

-PAS domain

-(GAF domain)

-Globin domain

J. Biol. Inorg. Chem. 8, 1 (2003)

Mechanisms of ligand discrimination

by heme proteins

PAS domain

Fig. 3 A Structure of the heme domain

of BjFixL. The FG loop is

shown in green. B Comparison of the

structure of FG loop and

conformation of Arg220 in the

unliganded ‘‘on’’ (blue) and

liganded ‘‘off’’ (tan) state [32, 33]

8

3’, 5’-cyclic AMP 5’-AMP

Ec DOS (Fe2+)

Function of Ec DOS Escherichia coli Direct Oxygen Sensor (Ec DOS) Gilles-Gonzales, M. A. et al. (2000)

Ec DOS (Fe3+)

Heme redox state regulates the function.

CO and NO inhibit catalysis.

9

Fe Fe

<Full-length>

Fe Fe

<PAS-A domain>

Tetramer Dimer

Oligomerization of Full-length and PAS-A

Fe

PAS-A PAS-B Phosphodiesterase

10

Fe (III) → Fe (II)

Ligand: H2O → Met95 Global structural change of FG-loop

11

Structure of Eｃ DOS PAS

N N

N N

O

O

O

O

N N

N N

O

O

O

O

Fe Fe

OHー

proximal side

distal side

Fe(II) complex Fe(III) complex

FG loop is rigid

Active

FG loop is flexible

Inactive

Met95

His77 His77

FG loop FG loop

12

Redox-induced scissor-type subunit motion of Ec DOSH

13

Domains Responsible for Oligomerization

100 247 401 491 605 705 807

dimerization tetramerization

100 247

147 Fe

21 84 144 201 336 799 807

PAS-A PAS-B phosphodiesterase

401 491

605 705

WT

PAS-A

DPAS-B

D A B C D

Oligo. Heme Catal.

4 O O

2 O X

4 X O

4 X O 4 O X 1 O X 4 O O 1 O X

148

Catalysis

Heme X

Tetramer O

14

Activation of Wild Type by Isolated Heme-PAS-A

Fe3+

PDE e-

PDE +PAS-A

activity: <1 activity: 5

PDE

activity: >25

Fe2+

PAS-B

PAS-A

PAS-B

PAS-A

PAS-B

PAS-A

Fe2+

+PAS-A +H77A PAS-A

PDE

Fe2+

PAS-B

PAS-A

Fe3+ Fe3+ apo

PDE

Fe2+

PAS-B

PAS-A apo

activity: 5

activity: 5

15

Protein microarray

Tissues

Cells

Body fluids

Total protein

Protein functional analysis

Protein quantification analysis

Genomics Proteomics

Comprehensive analysis of cellular proteins

Overview of the protein microarray technology

Protein microarray

method

16

Anti-(His)6 tag

Kd = 10-7-10-10 M

The extra peptide

tightly binds to

its antibody (Y).

(His)6-tagged Ec DOS: Extra peptide attached to the N-terminal

(a)： His-tag（extra peptide）of the protein tightly binds to its antibody on the

plate. Protein freedom and sensitivity substantially improved.

Detection of more natural protein-protein interaction is possible.

Development of the novel ultrasensitive protein microarray.

17

mAB, Fab fragment of monoclonal antibody against (His)6 tag

Cy5, cyanine5 (FITC, fluorescein isothiocyanate)

Upper: Interaction between His-tag and its antibody enhances the sensitivity. More freedom.

Lower: No interaction between His-tag and its antibody. Low freedom. Low sensitivity.

18

1 mg/ml Ec DOS

Negative Control

(a) Cy5 labeled PAS fragment Fe2+

Neg

ativ

e

20

0 µ

g/m

l

40

0 µ

g/m

l

60

0 µ

g/m

l

80

0 µ

g/m

l

10

00

µg/m

l

Reduced Ec DOS Fe2+

Oxidized Ec DOS Fe3+

1 mg/ml Ec DOS

Negative Control

(b) Cy5 labeled PAS fragment Fe3+

Neg

ativ

e

20

0 µ

g/m

l

40

0 µ

g/m

l

60

0 µ

g/m

l

80

0 µ

g/m

l

10

00

µg/m

l (a) (b)

1 mg/ml Ec DOS

Negative Control

(c ) Cy5 labeled PAS fragment

Neg

ativ

e

20

0 µ

g/m

l

40

0 µ

g/m

l

60

0 µ

g/m

l

80

0 µ

g/m

l

10

00

µg/m

l

(c) + K3Fe(CN)6

IC50 = 30 M

(a) Fe2+: High protein-protein interaction.

(b) Fe3+: Low protein-protein interaction.

(c): Oxidizing agent added to (b). No interaction

Catalytic activity is associated with

protein-protein interaction

19

+Substrate: High interaction

+Inhibitor: Low interaction

Inactive mutants: Low

interaction

20

The novel protein microarray proved that the catalytic activity of Ec DOS is closely associated with the protein-

protein interaction.

21

About the Cover

Protein Microarray System for

Detecting Protein-Protein Interactions

Using an Anti-His-Tag Antibody and Fluorescence Scanning

November 15, 2004 / Volume 76 / Issue 22 ------------------------------------------------

Art director Julie Farrar overlaid the structure of

heme with some "stop light" images created to resemble the authors' results.

Print || Close Window || Read this Article

Theoretically, detection of

10 fg protein is feasible.

Cover Art of Analytical Chemistry

22

1). Sasakura, Y. et al. (2002) J. Biol. Chem. 277, 2382.

2). Sato, A. et al. (2002) J. Biol. Chem. 277, 32650.

3). Yoshimura, T. et al. (2003) J. Biol. Chem. 278, 53105.

4). Taguchi, S. et al. (2004) J. Biol. Chem. 279, 3340.

5). Kurokawa, H. et al. (2004) J. Biol. Chem. 279, 20186.

The novel ultrasensitive protein microarray and its application

6). Sasakura, Y. et al. (2004) Anal. Chem. 76, 6521. Cover art

7). Sasakura, Y. et al. (2005) Biochemistry 44, 9598.

8). Sasakura, Y. et al. (2005) Acc. Chem. Res. 39, 37.

1.Profound protein structural changes occur upon heme redox change. 2.Isolated heme-bound PAS domain functions. 3.Protein-protein interaction is associated with catalysis.

23

Knockout E. coli Growth、Development、Differentiation、cAMP

Physiological Role of Ec DOS?

Turnover 0.1 min-1 toward cAMP c-di-GMP?

24 Knockout of Ec DOS caused cell filamentation.

aerobic growth

native W3110 Ddos W3110 native BL21 (DE3) Ddos BL21 (DE3)

Constructed Ec DOS -knockout E. coli (Ddos)

anaerobic growth

native W3110 Ddos W3110

Bar = 10 m

25

native W3110 (x1 diluted)

27.38 f mol / well

Ddos W3110 (x10 diluted)

46.14 f mol / well

Intracellular cAMP level in Ddos and native W3110

Knockout of Ec DOS caused excess

intracellular cAMP. filamentation

46.14 x 10 / 27.38 17

26

Input signals and output of c-di-GMP metabolism

Ute Römling et al., Molecular Microbiology, 2005, 57, 629–639

↓ ↓

27

Function of Ec DOS

PAS domain

O2, CO, NO

Sensor

domain

Effector domain Signal

transmitter

Fe2+

PDE domain

N

NHN

N

O

NH2O

OH

O

P

O

O--O

OP

O

O-

ON

N

N

HN

O

H2N O

OHHON

NHN

N

O

NH2O

OH

O

P

O-

OO

OP

O

O-

ON

N

N

HN

O

H2N O

HO

c-di-GMP l-di-GMP

Ec DOS

Fe (II)

Fe (II)

Fe (II)

Fe (II)

O2

O2 O2

Inactive Forms Active Forms

O2

DCSs

Ec DOS

AxPDEA1

FixL

DevS, DosT

Heme domain

of Ec DOS

Heme domain

of Ec DOS

Functional domain

Active

Functional domain

Inactive

O2

O2

Cover Art

Fe

Fe

Arg97

Met95

Fe(II)-O2

PDB ID: 1VB6

Arg97

Met95

Fe(II)

PDB ID: 1V9Z

Init

ial v

elo

city

of

the

PD

E

reac

tio

n (

min

-1)

M95A M95L M95H

Catalytic activities of the Fe(II) Met95 mutants

Fe(II) M95A and M95L: gas-independent. Fe(II) WT, M95H and Arg97: gas-dependent. Thus, Met95 plays a critical role in catalytic regulation.

WT

Tanaka et al. J. Biol. Chem. 282, 21301 (2007).

Fe(II) 、及び Fe(II)O2体の結晶構造

Fe(II) Fe(II)O2

Met95がヘムから脱離することでロック解除 J. Biol. Chem. 282, 21301 (2007), J. Am. Chem. Soc. 129, 3556 (2007)

1). Sasakura, Y. et al. (2002) J. Biol. Chem. 277, 2382.

2). Sato, A. et al. (2002) J. Biol. Chem. 277, 32650.

3). Yoshimura, T. et al. (2003) J. Biol. Chem. 278, 53105.

4). Taguchi, S. et al. (2004) J. Biol. Chem. 279, 3340.

5). Kurokawa, H. et al. (2004) J. Biol. Chem. 279, 20186.

6). El-Mashtoly, S. F. et al. (2007) J. Am. Chem. Soc. 129, 3556.

7). Tanaka, A., et al. (2007) J. Biol. Chem. 282, 21301.

8). El-Mashtoly, S. F. et al. (2008) J. Biol. Chem. 283, 19000.

33

33

dos (yddU) yddV

yddV and dos are organized as a bicistronic operon.

2 GTP DGC PDE

pGpG

DGC: Diguanylate cyclase PDE: Phosphodiesterase

signal signal

c-di-

GMP

YddV DOS

biofil

m

motilit

y

c-di-GMP metabolism in E. coli

Function of YddV

Sensor domain

GGDEF sequence

O2？

Signal DGC domain

Globin

fold

YddV-heme

Fe2+

2GTP c-di-GMP

35

8x104

6

4

2

0

A2

54

171615141312

Retention time (min)

1 h

2

3

5

71

2

3

5

7 h

GTP c-di-GMPFe(III)

6

5

4

3

2

1

0

µm

ol c

-di-

GM

P / µ

mo

l Y

dd

V

100806040200

Time (min)

Fe(II)-O2, Fe(II)-CO

Fe(III)

Fe(II)

v0 (min-1)

Fe(III)

Fe(II)-O2, Fe(II)-CO

Fe(II)

0.066 active

0.022 semi-active

0 inactive

Diguanylate cyclase activity

Fe (II)

Fe (II)

Fe (II)

Fe (II)

O2

O2 O2

Inactive Forms Active Forms

O2

YddV

Ec DOS

AxPDEA1

FixL

DevS, DosT

Heme domain

of GCS

Heme domain

of GCS

Functional domain

Active

Functional domain

Inactive

O2

O2

Cover Art

Fe

Fe

38

8x104

6

4

2

0

A2

54

16141210864

Retention time (min)

GTP

pGpG

1 h

3

5

8

GMP

GDP

200

150

100

50

0

[nu

cle

oti

de]

(µM

)

5004003002001000

Time (min)

pGpG GTP GMP

GTP

Fe(III) YddV

DGC reaction is rate-determining step.

0.066 min-1

Fe(III) DOS Fe(III) DOS

GMP c-di-GMP pGpG

8.1 min-1

Coupling reaction by YddV and DOS

39

H

O

Fe2+

His98

Fe2+

His98

Fe3+

His98

Inactive

Semi-Active

(Intermediate)

Active

Stable

0.00 min-1

0.022 min-1 0.066 min-1

+ O2

autooxidation

O

O

Tyr43

e-

O

N

H

H

Gln60

Activation mechanism triggered by O2

O2-

Biochemistry 49, 10381 (2010).