Ec DOS: Escherichia coli Direct Oxygen Sensor

Transcription

1 Ec DS: Escherichia coli Direct xygen Sensor 1

2 Heme-based gas sensor protein Signal (2,, C etc.) or redox state FixL, HemAT, sgc CooA, Ec DS Sensor domain (Heme) Functional domain Protein structural change Sensor domain (Heme) Functional domain Regulation of catalysis and transcription 2

3 Heme-based gas sensor proteins FixL 2 ass/disso His kinase/nitrogen fixation Ec DS Heme redox, 2 Phosphodiesterase HemAT 2 ass/disso Methylation/aerotaxis regulation sgc ass/disso Guanylate cyclase/smooth muscle viagla CooA C ass/disso Transcription regulation PAS2 C ass/disso Transcription regulation CBS C ass/disso Cystathionine -synthase 3

4 2 Sensor 2 Sensor 2 Sensor C Sensor Sensor C Sensor 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.

5 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 6

7 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 Function of Ec DS Escherichia coli Direct xygen Sensor (Ec DS) Gilles-Gonzales, M. A. et al. (2000) C and inhibit catalysis. Ec DS (Fe 2+ ) Ec DS (Fe 3+ ) 3, 5 -cyclic AMP 5 -AMP Heme redox state regulates the function. 8

9 ligomerization of Full-length and PAS-A PAS-A PAS-B Phosphodiesterase Fe Tetramer Dimer Fe Fe Fe Fe <Full-length> <PAS-A domain> 9

10 Fe (III) Fe (II) Ligand: H 2 Met95 Global structural change of FG-loop 10

11 Structure of Ec DS PAS Fe(II) complex FG loop Fe(III) complex FG loop Met95 distal side H ー Fe Fe His77 proximal side His77 FG loop is rigid Active FG loop is flexible Inactive 11

12 Redox-induced scissor-type subunit motion of Ec DSH 12

13 Domains Responsible for ligomerization PAS-A PAS-B Fe dimerization phosphodiesterase WT PAS-A tetramerization DPAS-B D A B C D ligo. Heme Catal. 4 2 X 4 X 4 X 4 X 1 X 4 1 X Catalysis Heme X Tetramer 13

14 Activation of Wild Type by Isolated Heme-PAS-A PAS-A PAS-A PAS-A Fe 3+ Fe 2+ PAS-B PDE e - PAS-B PDE activity: <1 activity: 5 +PAS-A Fe 2+ PAS-B PDE activity: >25 apo +H77A PAS-A apo PAS-A Fe 2+ PAS-B PDE activity: 5 +PAS-A Fe 3+ Fe 3+ PAS-A Fe 2+ PAS-B PDE activity: 5 14

15 method Protein microarray Genomics Proteomics Comprehensive analysis of cellular proteins verview of the protein microarray technology Tissues Cells Body fluids Total protein Protein microarray Protein functional analysis Protein quantification analysis 15

16 (His) 6 -tagged Ec DS: Extra peptide attached to the -terminal Anti-(His) 6 tag K d = M The extra peptide tightly binds to its antibody (Y). (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. 16

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: o interaction between His-tag and its antibody. Low freedom. Low sensitivity. 17

18 egative 200 µg/ml 400 µg/ml 600 µg/ml 800 µg/ml 1000 µg/ml egative 200 µg/ml 400 µg/ml 600 µg/ml 800 µg/ml 1000 µg/ml egative 200 µg/ml 400 µg/ml 600 µg/ml 800 µg/ml 1000 µg/ml (a) Cy5 labeled PAS fragment Fe2+ (b) Cy5 labeled PAS fragment Fe3+ (a) (b) 1 mg/ml Ec DS egative Control 1 mg/ml Ec DS egative Control Reduced Ec DS Fe2+ xidized Ec DS Fe3+ (c ) Cy5 labeled PAS fragment 1 mg/ml Ec DS egative Control (c) + K3Fe(C)6 IC 50 = 30 M (a) Fe 2+ : High protein-protein interaction. (b) Fe 3+ : Low protein-protein interaction. (c): xidizing agent added to (b). o interaction Catalytic activity is associated with 18 protein-protein interaction

19 +Substrate: High interaction +Inhibitor: Low interaction Inactive mutants: Low interaction 19

20 The novel protein microarray proved that the catalytic activity of Ec DS is closely associated with the proteinprotein interaction. 20

21 Cover Art of Analytical Chemistry About the Cover Protein Microarray System for Detecting Protein-Protein Interactions Using an Anti-His-Tag Antibody and Fluorescence Scanning ovember 15, 2004 / Volume 76 / Issue 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. 21

22 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. 1). Sasakura, Y. et al. (2002) J. Biol. Chem. 277, ). Sato, A. et al. (2002) J. Biol. Chem. 277, ). Yoshimura, T. et al. (2003) J. Biol. Chem. 278, ). Taguchi, S. et al. (2004) J. Biol. Chem. 279, ). Kurokawa, H. et al. (2004) J. Biol. Chem. 279, The novel ultrasensitive protein microarray and its application 6). Sasakura, Y. et al. (2004) Anal. Chem. 76, Cover art 7). Sasakura, Y. et al. (2005) Biochemistry 44, ). Sasakura, Y. et al. (2005) Acc. Chem. Res. 39,

23 Physiological Role of Ec DS? Turnover 0.1 min -1 toward camp c-di-gmp? Knockout E. coli Growth Development Differentiation camp 23

24 Constructed Ec DS -knockout E. coli (Ddos) aerobic growth native W3110 Ddos W3110 native BL21 (DE3) Ddos BL21 (DE3) anaerobic growth native W3110 Ddos W3110 Bar = 10 m Knockout of Ec DS caused cell filamentation. 24

25 Intracellular camp level in Ddos and native W3110 native W3110 (x1 diluted) f mol / well Ddos W3110 (x10 diluted) f mol / well x 10 / Knockout of Ec DS caused excess intracellular camp. filamentation 25

26 Input signals and output of c-di-gmp metabolism Ute Römling et al., Molecular Microbiology, 2005, 57,

27 Function of Ec DS 2, C, Signal transmitter PAS domain Fe 2+ Sensor domain PDE domain Effector domain H 2 H H - P P - c-di-gmp H H H 2 Ec DS H 2 H H H - P P - - H l-di-gmp H H 2 27

28 2 2 Fe (II) Fe (II) DCSs Ec DS 2 Fe (II) 2 Fe (II) AxPDEA1 FixL DevS, DosT Inactive Forms Active Forms

29 Functional domain Inactive Functional domain Active 2 2 Fe Fe Heme domain of Ec DS Heme domain of Ec DS

30 Initial velocity of the PDE reaction (min -1 ) Catalytic activities of the Fe(II) Met95 mutants Fe(II) Arg97 Met95 PDB ID: 1V9Z Fe(II)- 2 Met95 Arg97 WT M95A M95L M95H Fe(II) M95A and M95L: gas-independent. Fe(II) WT, M95H and Arg97: gas-dependent. Thus, Met95 plays a critical role in catalytic regulation. PDB ID: 1VB6 Tanaka et al. J. Biol. Chem. 282, (2007).

31 Fe(II) 及び Fe(II) 2 体の結晶構造 Fe(II) Fe(II) 2 Met95 がヘムから脱離することでロック解除 J. Biol. Chem. 282, (2007), J. Am. Chem. Soc. 129, 3556 (200

32 1). Sasakura, Y. et al. (2002) J. Biol. Chem. 277, ). Sato, A. et al. (2002) J. Biol. Chem. 277, ). Yoshimura, T. et al. (2003) J. Biol. Chem. 278, ). Taguchi, S. et al. (2004) J. Biol. Chem. 279, ). Kurokawa, H. et al. (2004) J. Biol. Chem. 279, ). El-Mashtoly, S. F. et al. (2007) J. Am. Chem. Soc. 129, ). Tanaka, A., et al. (2007) J. Biol. Chem. 282, ). El-Mashtoly, S. F. et al. (2008) J. Biol. Chem. 283,

33 c-di-gmp metabolism in E. coli dos (yddu) yddv yddv and dos are organized as a bicistronic operon. DGC: Diguanylate cyclase signal PDE: Phosphodiesterase signal 2 GTP YddV DGC PDE DS pgpg biofil c-di- GMP motilit 33 33

34 Function of YddV 2? Signal Sensor domain YddV-heme Globin fold Fe 2+ GGDEF sequence DGC domain 2GTP c-di-gmp

35 A254 µmol c-di-gmp / µmol YddV Fe(III) 8x GTP 1 h Diguanylate cyclase activity Retention time (min) c-di-gmp 6 7 h Fe(III) Fe(II)- 2, Fe(II)-C Fe(II) Time (min) Fe(III) Fe(II)- 2, Fe(II)-C Fe(II) v 0 (min -1 ) active semi-active 0 inactive 35

36 2 2 Fe (II) Fe (II) YddV Ec DS 2 Fe (II) 2 Fe (II) AxPDEA1 FixL DevS, DosT Inactive Forms Active Forms

37 Functional domain Inactive Functional domain Active 2 2 Fe Fe Heme domain of GCS Heme domain of GCS

38 A254 [nucleotide] (µm) Coupling reaction by YddV and DS Fe(III) YddV Fe(III) DS Fe(III) DS GTP c-di-gmp pgpg GMP min min -1 8x GMP 1 h 3 GTP pgpg GTP GMP 4 2 GDP 5 8 pgpg Retention time (min) Time (min) DGC reaction is rate-determining step. 38

39 Activation mechanism triggered by min -1 Inactive Tyr43 Fe Gln60 e - His H H H Fe 3+ Fe 2+ His98 autooxidation His min-1 Active Stable Semi-Active (Intermediate) min -1 Biochemistry 49, (2010). 39