Tsr red AW blue

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Cryo-tomography
Zhang et al., PNAS 2007
Tsr red A-W blue
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(No Transcript)
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Gain
Change in rotational bias/ Change in receptor
occupancy 60
Dennis Bray, PNAS, 2002
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2-D Hexagonal network
A 36-fold gain at the receptors indicates that
one receptor molecule can control the activity
of three dozen kinase molecules, implying a
functional network that links one receptor to
multiple copies of the kinase. The response to
attractant stimulation is cooperative Hill
coefficients as high as 10 have been observed
.These properties imply that receptors operate as
allosteric arrays with as many as several dozen
in the cooperative unit.
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Adaptation compartment
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Swimming bacteria sense spatial gradients by
detecting concentrations over time
In deciding whether to continue running in a
favorable direction, the chemosensory system
compares a measure of current concentration to a
record of the just-previous concentration
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Background
Tar/Tsr
CheR
NWETF
NWETF is absent in low abundance receptors Trg
and Tap
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CheBP is a methylesterase and also an deamidase
converts Q to E
Q can be converted to E either naturally by CheB,
or artificially by mutagenesis
Amides at methyl-accepting sites are in large
part the functional equivalents of methylesters
QQQQ form is highly active in ternary complex in
vitro, and promotes tumbling in vivo EEEE
form is inactive in ternary complex formation in
vitro, and suppresses tumbling in vivo The wild
type receptor, i.e. the QEQE form, has
intermediate activities both in vivo and in
vitro
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Low abundance receptors (Trg/Tap) mediate a
strong response to their signals in wild-type
cells, but stimulate kinase only weakly in vitro
These receptors cannot support chemotaxis when
expressed alone Alone, they are defective in
methylation, and lack a binding site for CheR
This suggests that proximity of high and low
abundance receptors may promote inter-dimer
methylation
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Inter-dimer Methylation Model
Trg/Tap
Tsr/Tar
Shaded area on CheR active site
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Does receptor methylation take place via an
inter-dimer process?
(Remember, these experiments were done before
discovery of trimer-of-dimers)
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Experimental Design
Tsr missing NWETF but having methylatable Es
Substrate subunits Tsr missing methylatable Es
but having NWETF Binding subunits Mix and
test activity
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The SH groups can be crosslinked with oxidizing
agents
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D36C/DC34 4Q
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Reconstituted membrane system with Tsr/CheW/CheA
CheY Phosphorylation level
Result C-terminal truncation is functional in
CheA phosphotransferase activity.
Amides at methyl-accepting sites are in large
part the functional equivalents of methylesters
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Tsr methylation by CheR
WT
Coexpression
DC34 4Q
Controls Substrate- Subunit- Membrane Mixt. of
the two
Why is the Me level in co-expressed subunits less
than WT?
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Lanes 3,4 Cross-linking does not affect
Me Lanes 7,8 Only homodimers will X-link
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Serine stimuli increase inter-subunit methylation
rate
1. Which data support inter-subunit methylation
upon serine stimulus?
2. Do the data rule out intra-subunit methylation?
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Conclusions
Inter-dimer methylation occurs efficiently Data
support the idea of receptor clustering
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What controls the rates of methylation and
demethylation?
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Each dimer has a range of 8 possible -/0
values When all 8 are ve, kinase is
inactive When all 8 are neutral, kinase is
maximallu active The all state may be
repulsive and network may expand The all 0 state
may be interactive and network may
contract Thus, activities of CheB and CheR may
be critical to signaling
Jeff Stock
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Expansion and contraction of network may control
CheA
The kinase can promote direct physical coupling
between trimers by supporting tight clustering
(top), or promote indirect coupling by mediating
conformational spread (bottom).
Fluorescently tagged receptors - measure changes
in fluorescence anisotropy between fluorophores
on different dimers of a trimer within 3 sec,
attractants move these fluorophores farther
apart, and repellents move them closer together.
Vaknin Berg, JMB, 2007
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Tar/Tsr
NWETF
Efficient adaptational demethylation of
chemoreceptors requires the same enzyme-docking
site as efficient methylation
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1. The pentapeptide (PP) sequence is necessary
for the demethylation activity of CheB 2. CheB
binds to the PP in Tar, as does CheR. However,
their mechanism of activation is distinctly
different 3. For CheR, the PP acts as a
high-affinity docking site, enhancing methylation
by increasing enzyme concentration near the
Me-accepting glutamates 4. For CheB, interaction
with PP activates demethylation by allosterically
activating the receptor substrate, not the enzyme
(i.e. increases rate without increasing affinity)
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CheB and CheR are indeed at the pole as well
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A sense ational gathering!
CheY
CheZ
Bano et al., Mol. Micro. 2004
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Lipkow et al 2005 J. Bact
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Computer-generated simulation of distribution of
all molecules
CheYP is Red CheY is Black FliM is Blue
Lipkow et al 2005 J. Bact
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Paths of single molecules 100 msec in the life
of a CheY molecule
Comment by Hazelbauer, J. Bacteriol. 2005
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Summary We find that the time delay between
stimulus and response differs for flagellar
motors located at different positions in the
cell. We explore different possible locations
for the phosphatase CheZ and show conditions
under which a gradient of CheYp exists in the
cell. The behavior we report is in good agreement
with analytical solutions but goes far beyond
what would be possible to calculate
analytically. Moreover, the molecular details
revealed by our simulationssuch as changing
lifetimes of CheYp molecules or the responses of
motors at different locations in the cellexceed
the resolution of currently available techniques.
The introduction of inert blocks into the
cytoplasm, representing impenetrable structures
such as the nucleoid and large protein
complexes, produces a fall in the apparent
diffusion coefficient of CheYp and enhances the
differences between motors. These and other
results are left as predictions for future
experiments.
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• "Why do we care? What does it matter that the
  response of a tiny microbe has this or that
  numerical value?" The answer, surely, is that the
  chemotaxis pathway of E. coli is so well
  documented it has become, in a sense, the
  bellwether of signal transduction. If we are
  unable to understand how this simple network of
  proteins functions as an integrated system, then
  what hope have we of understanding the complex
  pathways in eukaryotic cells?

H. Berg E. coli demands our admiration and
respect
Dennis Bray, PNAS, 2002