Bacterial Signal Transduction

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Phosphorylation plays a dual role in activating
CheB
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Model for CheB activation
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Rec response regulator receiver contains the
phophorylated Asp Most response regulators have
a C-terminal DNA binding domain (HTH), but a
few, like CheB have enzymatic activity
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Che Y

• 5 a helices, 5 ß sheets

Santoro, J. J. Mol Biol (1995) 247(4), 717
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(No Transcript)
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Throwing the Switch
Silversmith Bourret, Trends Microbiol. 7,
16-22, 1999
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Zhu et al., 1997. Crystal structures of CheY
mutants Y106W and T87I/Y106W CheY activation
correlates with movement of residue 106. J. Biol.
Chem. 272 5000-5006.
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Question?
How does phosphorylation activate CheY? CheYD13K
causes CW rotation in absence of phosphorylation
(Asp 57 is the phosphorylated reside) This
paper compares structure of two mutants with
opposite switch behavior in order to answer the
question above
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Y106

• Position 106 of CheY is a conserved aromatic
  residue (Y or F) in the response regulator
  superfamily.
• - Y106 is located on the putative switch binding
  surface

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• Y106 is a rotamer whose side chain can occupy
  both the inside (solvent-inaccessible) and
  outside (solvent-exposed) position.
• - When the Y106 side chain is inside, the OH
  group forms a hydrogen bond with the O? of T-87
  through a solvent molecule.

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• In the T87I mutant which is unable to confer CW
  flagella rotation, Y106 side chain exclusively
  occupies the outside

T87I is smooth-swimming and hence non-chemotactic
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• Hypothesis movement of the side chain of residue
  106 modulates the activation state of CheY.

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They know that T87I causes CCW behavior and that
Y106 is outside They have a mutant Y106W that
causes CW behavior Based on what they know,
they predict Y106W to be inside. When they
create a double mutant Y106W/T87I, they
find CheY-Y106W/T87I is inactive CCW
rotation So, both mutants have Y106W but show
opposite behavior Does behavior correlate with
structure?
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Methods

• Behavioral assays chemotaxis and motor rotation

• Protein Phosphorylation assay as in Hess et al

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Pfeffer assay Congregation of bacteria at mouth
of capillary containing meat extract
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Chemotaxis behavior of mutants
spectrophotometric assay
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Motor behavior of mutants
Tethering assay
http//www.rowland.org/labs/bacteria/movies_tether
ed.html
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Cells in a cloned population of coliform bacteria
exhibit a wide range of swimming behaviors, a
form of non-genetic individuality. We used
computer models to examine the proposition that
these variations are due to differences in the
number of chemotaxis signaling molecules from
one cell to the next. Simulations were run in
which the concentrations of seven gene products
in the chemotaxis pathway were changed either
deterministically or stochastically, with the
changes derived from independent normal
distributions. Computer models with two
adaptation mechanisms were compared with
experimental results from observations on
individuals drawn from genetically identical
populations. The range of swimming
behavior predicted for cells with a standard
deviation of protein copy number per cell of 10
of the mean was found to match closely the
experimental range of the wild-type population.
Levin et al., Biophys J, 74, 175-181, 1998
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• Are the smooth-swimming phenotypes of the T78I
  and T87I/Y106W due to defects in phosphorylation?

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in vitro phosphorylation of mutants
Stability of CheP
Behavior not correlated with phosphorylation
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The structural differences at position 106
WT
T87I
CCW?
CW?
CCW?
Behavior correlated with position of Y106
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Conclusion
The signaling ability of CheY correlates with the
position of residue 106
What is the role of phosphorylation?
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• Due to the short half life of cheY-P, NMR
  analysis had been used to identify chemical
  shifts in regions of cheY/cheY-P.

• Global changes pose a problem in that structural
  information alone does not identify function in a
  particular response regulator.

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How is the Y106 position related to the CCW to
CW transition?
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Interaction with FliM
1. Different amino acid substitutions at the 106
position bind FliM like wild-type 106 not likely
to be directly involved in binding 2. D13K is
non-phophorylatable, signals constitutively, but
does not show increased binding 3. Conversely,
CheY87TI cannot signal but binds FliM like
wild-type
Binding is necessary but not sufficient. A
post-binding event is likely responsible for
switching
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Two models for the physical mechanism of throwing
the switch
Deterministic model a sequential model of
binding and switching
indicates positive effect of the protein at
each step
Both CheY95!V and CheY106YW show CW behavior
cheY95IV has a higher affinity for FliM
cheY106YW does not have a higher affinity for FliM
Silversmith Bourret, Trends Microbiol. 7,
16-22, 1999
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The Stochastic model binding of CheY-P to the
switch merely increase the probability of a CW
rotation
CCW and CW represent thermodynamic states that
are in equilibrium and are capable of switching
at a small but finite rate, even in the absence
of CheY.
Silversmith Bourret, Trends Microbiol. 7,
16-22, 1999
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Model for CheB activation

• CheB can exist in two conformations - a closed,
  less active state and an open, more active state.
  Phosphorylation shifts the equilibrium to the
  open state