Sensing Mechanism of Globin-coupled Oxygen Sensor AfGcHK

Transcription

1 Sensing Mechanism of Globin-coupled Oxygen Sensor AfGcHK 1

2 Two-component signal transduction system (1) Signal redox, temperature, osmolarity etc. sensor (2) Autophospholylation ATP HK His - P ADP (HK) (3) Phosphotransfer Asp Rec - P output Response regulator (RR) (4) Output Regulation of catalytic activity, transcription, or chemotaxisis 2

3 Heme-regulated histidine kinases Gas sensor Effector FixL PAS PAS O 2 DosS (DevS) DosT GAF GAF O 2, NO H-NOX H-NOX NO Heme sensor ChrS TM heme 3

4 Globin-coupled oxygen sensors Mammalian globin Sensor globin SW Mb globin HemAT-Bs globin MCP methyl-accepting chemotaxis protein Zhang, W., and Phillips, G. N. Jr. (2003) Structure 11,

5 Heme-regulated histidine kinases Gas sensor Effector GcHK??? globin O 2 FixL PAS PAS O 2 DosS (DevS) DosT GAF GAF O 2, NO H-NOX H-NOX NO Heme sensor ChrS TM heme 5

6 Identification of the novel globin-coupled histidine kinase Anaeromixobacter sp. Fw109-5 (HK) Anae109_2438 globin 382 aa His183 Anae109_2439 Asp52 Rec Asp169 Rec Response regulator (RR) 238 aa Novel HK and RR were identified in the Anaeromixobacter sp. Fw109-5 genome. 6

7 Characterization of the globin-coupled histidine kinase (1) Signal O 2 heme globin (2) Structural changes & signal transduction histidine kinase ATP His183 - P Objectives ADP 382 aa (3) Autophospholylation 1. Characterization of the novel globin-coupled histidine kinase, AfGcHK. 2. Elucidation of the O 2 recognition mechanism. 7

8 Absorbance Absorption spectra Absorbance (III) (II) (II)-CO (II)-O 2 (II)-NO x x Wav elength (nm) Wav elength (nm) AfGcHK (III) (II) (II)-O 2 (II)-CO (II)-NO 411, , , 545, , 541, , 548, 567 6cLS 5cHS 6cLS 6cLS 6cLS SW Mb 410, 505, , , 543, , 542, , 548, 579 6cHS 5cHS 6cLS 6cLS 6cLS (II)-O2 Half life > 3 days HS: High-spin, LS: Low-spin (nm) 8

9 Principle of Mn 2+ -Phos-tag SDS-PAGE Time P-Protein Phos-tag TM AAL-107 Mw = Protein 9

10 % Phospholylation Time (III) (II) (II)-O 2 (II)-CO Autophospholylation activities (min) P-GcHK GcHK P-GcHK GcHK P-GcHK GcHK P-GcHK GcHK (III) (II) 2 (II)-O 2 (II)-CO Activity (III) (II)-O 2, (II)-CO 4 6 Time (min) (II) 8 10

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12 Absorbance Absorption spectra Absorbance Y45F (III) (II) (II)-O H99A x Wav elength (nm) Wav elength (nm) (III) (II) (II)-O 2 WT Y45L Y45F Y45W H99A 411, , , , 534 ND 431, , , , , 545, , 548, , 546, , 547, 580 H99A lost heme binding ability. (nm) 12

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14 Identification of an autophospholylation site (II)-O 2 Time WT H183A (min) P-GcHK GcHK H183A was not autophospholyated. Therefore, His183 is the autophospholylation site. 14

15 % Phosphorylation (A) WT Time (min) (B) WT (III) (II) (D) (II)-O 2 (C) H99A H183A 30 (II)-CO Y45F Time (min) 15 (II)-O 2 Heme is furnished to supress catalysis. 15

16 Phosphotransfer reaction (II)-O 2 AfGcHK RR His183 - P P P globin Rec Rec Time (min) P-P-RR P-RR RR P-GcHK GcHK RR was di-phosphorylated. Therefore, RR has two phosphorylation sites. 16

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18 Identification of phosphorylation sites Time WT D52A D169A D52A/ D169A (min) P-P-RR P-RR RR P-GcHK GcHK D52A and D169A were mono-phosphorylated. D52A/D169A was not phosphorylated. Therefore, Asp52 and Asp169 are the phosphorylation sites. 18

19 Summary 1 1. AfGcHK is the dimeric globin-coupled oxygen sensor histidine kinase. 2. His183 is the autophosphorylation site. 3. AfGcHK transfers two phosphoryl groups to its cognate RR. 4. Asp52 and Asp169 are the phospholylation sites. 19

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21 Absorbance Absorption spectra Absorbance Y45F (III) (II) (II)-O H99A x Wav elength (nm) Wav elength (nm) WT Y45L Y45F Y45W H99A (III) (II) (II)-O 2 (II)-CO 411, , , 545, , 541, , , , 548, , 541, , , , 546, , 541, , , , 547, , 543, 565 ND H99A lost heme binding ability. (nm) 21

22 Absorbance O 2 dissociation rate constants WT (II)-O 2 (II) O dithionite His99 k off dissociation His99 A Wav elength (nm) 550 WT Y45F Y45W WT Y45L Y45F Y45W H183A k off (s -1 ) Time (s) HemAT-Bs SW Mb

23 O 2 ligand binding parameters k on (µm -1 s -1 ) k off (s -1 ) K d (µm) AfGcHK WT 1.3, , 0.67 Y45L 1.5, , 100 Y45F 1.8, , 2.3 Y45W 1.3, , 28 YddV BpeGReg Ec DOS Bj FixL HemAT-Bs SW Mb K d value was calculated from k on and k off values. 23

24 Intensity Resonance Raman spectra n -O2 n O-O (II)-O 2 l ex = nm Tyr45 16 O 2-18 O 2 WT? O O H? O Y45F 2+ His Raman Shift (cm -1 ) Raman Shift (cm -1 ) n -O2 n O-O WT Y45F (cm -1 ) Tyr45 forms a hydrogen bond(s) with O 2. 24

25 Summary 2 1. Tyr45 is important for stabilization of the (II)-O 2 complex. 2. Tyr45 forms a hydrogen bond with O 2. 25

26 Summary Tyr45 Tyr45 OH Tyr45 O H OH e - + O 2 O H O? O His99 His99 His99 6cLS 5cHS 6cLS active inactive active & stable (II)-O2 Half life > 3 days K d O , 0.67 µm 26

27 Summary O 2 AfGcHK Anae109_2438 globin His183 - P (HK) 382 aa ATP ADP RR Anae109_2439 Asp52 Rec - P Asp169 Rec - P Response regulator (RR) 238 aa Regulation of unknown physiological process Slime bacteria Anaeromixobacter sp. Fw109-5 Anaeromixobacter dehalogenans Myxococcus xanthus 27

28 Heme-regulated histidine kinases Gas sensor Effector AfGcHK globin O 2 FixL PAS PAS O 2 DosS (DevS) DosT GAF GAF O 2, NO H-NOX H-NOX NO Heme sensor ChrS TM heme 28

29 FIGURE 1. Alignment of heme-bound oxygen-sensing and functional domains of the heme-based oxygen sensors. Left, the C-terminal HK domain in the two-component system containing N-terminal heme-bound globin, PAS, or GAF domains. Right, the C-terminal functional domains associated with MCP, c-digmp homeostasis (DGC, synthesis; and PDE, degradation), and unknown functions (transmembrane (TM)) containing the N-terminal hemebound globin or PAS domain. GCSs are defined as chimeric proteins composed of heme-bound globin and functional domains (14 18). Note that EcDOS and FixL have two tandem PAS domains; the heme iron complex is bound to the first PAS domain in EcDOSand to the second PAS domain in FixL. Similarly, DevS and DosT have two tandem GAF domains; in both cases, the heme iron complex is bound to the first GAF domain. Note that globin folds are not always localized at the N terminus but are inserted into diverse regions, as predicted from the amino acid sequences of sensor globins that are yet to be characterized (14 18). AxPDEA1, A. xylinum PDEA1. J. Biol. Chem. 288, (2013) 29