Molecular Structure of CheAD289

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Plasmid combination ? Wild-type ? pRBB16-R73K
pMK113-258oc ? pRBB16-R73K pMK113-T154I ?
pRBB16-R73K pMK113-?tar
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Hess, J. F. et al., 1988. Phosphorylation of
three proteins in the signaling pathway of
bacterial  chemotaxis. Cell 53 79-87. Bilwes et
al., 1999. Structure of CheA, a
signal- Transducing histidine kinase. Cell
96131-141.
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The Che Proteins
What they know There are six cytoplasmic che
gene products required for signal transduction
in bacterial chemotaxis Four cytoplasmic
proteins in the excitation response? CheA, CheW,
CheY, and CheZ Two cytoplasmic proteins in the
adaptation response ? CheR and CheB
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What they suspect A signal relay based on
phospho transfer, because of homologies between
CheA and another kinase (NtrB) that is involved
in signal transduction.
CheY/B
CheA
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Question?
Is phosphate transfer the chemical currency of
signaling through the chemotaxis system?
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The phosphotransfer reaction of 2-component
systems
(CheA)
(CheY/CheB)
(CheZ)
Mg
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CheA is autophosphorylated
Fig. 1
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Optimization of Rates of Autophosphorylation
Fig.2
Top optimized reaction with 50mM KCl 0.1mM
g-32PATP 5mM MgCl2 at pH 8.5 Bottom
previous unoptimized conditions
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Dephosphorylation of CheA by B,Y,Z

• Phosphorylated CheA was found to be relatively
  stable ? a radiolabeled phosphorylated CheA could
  be prepared and frozen without significant
  hydrolysis of the phosphoryl group. So they used
  this as their phosphodonor

What does this table tell us about the possible
functions of CheY, CheB and CheZ?
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Figures 3-4
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CheZ Accelerates the Dephosphorylation of
CheA(Only in the Presence of CheY)
Figure 5
A CheAP Y B CheAP, CheY, and CheZ in a
1011 molar ratio C CheAP, CheY, and CheZ in a
1010.1 molar ratio D CheAP and CheZ in a 101
molar ratio
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CheA-P and CheY in a 101 molar ratio CheA-P,
CheY and CheZ in 1011 molar ratio CheA-P, CheY
and CheZ in 1010.1 molar ratio
CheZ does not accelerate dephos of CheBP
Figure 6
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Conclusions

• CheA becomes autophosphorylated in the presence
  of ATP
• CheAP can transfer its phosphate to CheB or CheY
  
• CheZ accelerates the hydrolysis of CheYP
• CheA is likely at the branch point for the
  dissemination of sensory information gathered by
  the receptor

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CheW
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Hess, J. F. et al., 1988. Phosphorylation of
three proteins in the signaling pathway of
bacterial  chemotaxis. Cell 53 79-87. Bilwes et
al., 1999. Structure of CheA, a
signal- Transducing histidine kinase. Cell
96131-141.
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CheA is a dimer in solution
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CheA autophosphorylates in trans
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CheA is autophosphorylated
Fig. 1
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CheA from Thermotoga maritima

• Attemps to crystallize CheA from E.coli failed.
• Che genes from T. maritima show homology to E.
  coli.
• The crystallized portion (CheAD289) lacks the two
  amino terminal
• domains P1 and P2

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Histidine Kinases

• All histidine kinases have 5 regions or boxes of
  amino acid sequence similarity. The ATP-binding
  domain has 4 regions of sequence similarity ? N,
  G1, F, and G2

Two major classes of histidine kinases ? depends
on position of the substrate histidine and
surrounding residues (H box) with respect to the
ATP-binding domain
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P1 has the phosphorylated His P2 binds
CheB/CheY P3 helps dimerize P4 is the ATP
binding kinase domain P5 is the regulatory
domain, binds CheW.
The crystal structure is for a fragment missing
P1-P2
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Molecular Structure of CheAD289

• Its a dimer.
• There are 3 distinct domains
• Kinase domain (residues 355-540)
• Dimerization domain (residues 290-354)
• Regulatory domain (residues 541-671)
• The two subunits in the dimer are not perfectly
  symmetrical

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The Overall CheAD289 Structure
Catalytic domain has homology to a superfamily of
ATPases e.g GyraseB
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Structure suggests mechanism
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Kinase Regulation (P5)
The Che A regulatory domain has similar topology
to SH3 (Src- homology-3) domains ? regulate
kinase activity in higher organisms
(protein-protein interactions)
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Conclusions
1. P3-P5 fragment is dimeric but not perfectly
symmetric 2. P3 is the dimerization domain 3.
P4 is the kinase domain Identification of
ATP-binding pocket 4 P5 likely interacts with
CheW has homology to eukaryotic SH3 domains 5.
Structure suggests mechanism of
trans-autophosphorylation
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