Post on 20-Dec-2015
transcript
Modeling the chemosensing system of E. coli
Ned Wingreen – Princeton
Juan Keymer – Princeton
Robert Endres – Princeton/NEC
Yigal Meir – Ben Gurion University
Outline• Introduction to chemotaxis in E. coli
– Runs and tumbles – Signaling properties– The chemotaxis network
• FRET – New probe of receptor activity– Two regimes of activity– Receptors function collectively
• Modeling• How does adaptation work?
Signaling properties of the chemotaxis network
• “Precise and robust adaptation”: range of 3-4 orders of magnitude of attractant
• “Signal integration”: multiple attractants
• “Sensitivity”: amplification
Segall, Block, and Berg (1986)
CCW vs CW bias for tethered cells in response to step in attractant
The chemotaxis networkht
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……but signal integration and sensitivity but signal integration and sensitivity are still not well understood.are still not well understood.
Chemoreceptors
Tar - aspartate, glutamate (~900 copies)
Tsr - serine (~1600)
Trg - ribose, galactose (~150)
Tap - dipeptides (~150)
(Aer - oxygen via FAD (150?))
Methyl binding sitesCheB, CheR
Sensor
Linker region
Cytoplasmicdomain
CheA / CheWbinding region
Stock (2000)
Dimer
•Attractant binding inhibits phosphorylation of CheA
•Adaptation:
More attractant → increased methylation by CheR → increased rate of phosphorylation of CheA
Less attractant → increased demethylation by CheB → decreased rate of phosphorylation of CheA
Transmembrane Transmembrane heliceshelices
380 A
Chemoreceptor clustering
Receptors are clustered globally into a large array, and locally into trimers of dimers.
Ge
stw
icki
G
est
wic
ki e
t al.
et a
l. (2
000
) (
200
0)
Kim Kim et al. et al. (1999); Studdert (1999); Studdert and Parkinson (2004)and Parkinson (2004)
In vivo FRET studies of receptor activity
Sourjik and Berg (2002)Sourjik and Berg (2002)
Real-time measurement of rate of phosphorylation of CheY.
FRET also allows subcellular imaging, Vaknin And Berg (2004).
Sourjik and Berg (2002)Regime I:• Activity moderate to low at zero ambient MeAsp (0.06,1)• KD small and almost constant
Regime II:• Activity high (saturated?) at zero ambient MeAsp (1.3-1.9)• KD1 large and increasing with methylation• Plateau in activity• KD2 approximately constant
FRET data: two regimes of activity
Regime I
Regime II
Two regimes of receptor activity consistent with 2-state receptor model.
• Originally proposed by Asakura and Honda (1984).• Modified by Barkai and Leibler (1998) to explain precise and
robust adaptation: – Receptor complex has 2 states: “on”, i.e. active as kinase,
and “off”, i.e. inactive as kinase.– Demethylation only occurs in “on” state, i.e. when receptor
is active, so that
– Therefore, at steady state,
– Which implies precise and robust adaptation of each receptor complex to a fixed activity.
2-state receptor model
Activity[CheB]-CheR][nMethylatio
badt
d
[CheB]/[CheR] Activity baBarkai and Leibler (1997)
Regime I:• Activity low to very low at zero ligand concentration • KD = KD
off
Regime II:• Activity high (saturated) at zero ligand concentration• KD increasing as εon ↓
offoffon
on
off
onActivity
eK
Cee
eP
D
Two regimes of a 2-state receptor
However, single receptor does not account for low apparent KD in Regime I.
On Off
Off
Regime I Regime II
On
Off
On
Off
Off Off
Ligand Ligand
Free Energy
But first, a “1-state” receptor:
Ligand
KD
DK
CP
1
1bound ligand no
On
offon
off
on ,11
1Activity
)(
N
K
C
D
NeP Regime I (Δε > 0):
• Low activity ~ e-NΔε at zero ligand concentration• KD = KD
off / N
• Hill coefficient = 1
Regime II (Δε < 0):• KD = KD
off e-Δε
• Hill coefficient = N
Receptor-receptor couplingDuke and Bray (1999)
Duke and Bray (1999) proposed that receptor-receptor coupling could enhance sensitivity to ligands.
Toy model: if N receptors are all “on” or all “off” together,
Receptor-receptor coupling gives enhanced sensitivity (low KD) in Regime I, and enhanced cooperativity (high Hill coefficient ) in Regime II.
Regime I• Low, constant KD
• Activity low at zero ligand concentration• Hill coefficient ≈ 1
Regime II:• KD1 increasing with methylation• Activity high at zero ligand concentration• Hill coefficient ≈ 1 ?• Plateau in activity ?
Review of FRET data
= KDoff
/N, value of N ?
Hill coefficient increases with receptor homogeneity.
Must consider mixture of different receptor types.
Sourjik and Berg (2004)
More Tars
Less Tars
Regime I: • KD set by coupling energy EJ
Regime II:• Plateaus: Tars “off”, Tsrs “on” • Hill coefficient ≈ 1, no cooperativity because Tar receptors separated by Tsr receptors
Model: 1d mixed lattice of receptorsN
orm
aliz
ed A
ctiv
ity
Log([MeAsp])
TsrTarEJ
Receptors are in Regime II:
• Hill coefficient increases with homogeneity because clusters of identical receptors grow.
• KD (or KD1) increases as lattice becomes more mixed because of coupling EJ to “on” receptors.
Receptor homogeneity and cooperativity
Nor
mal
ized
Act
ivity
Log([MeAsp])
More Tars
Less Tars
Adaptation
Adaptation uses methylation to return all receptors to Δε ≈ 0, and thereby enhances sensitivity.
TsrTar
Δε ≈ 0 Off
Δε > 0 Δε > 0 Δε > 0Δε ≈ 0 Δε ≈ 0 Δε ≈ 0
Scaling of wild-type response data
Sourjik and Berg: Δ[MeAsp] → ΔFRET{Tar(QEQE)}
Sou
rjik
and
Ber
g (2
002)
ΔFRET for Tar(QEQE) strain
“Free energy” scaling: Δ[MeAsp] → Δ(Fon – Foff)
Doesn’t collapse zero-ambient data.
Includes zero-ambient data!
Predictions
For homogeneous lattice –
• Transition from Regime I to Regime II with methylation
• Adaptation range set by KDon
Sourjik (unpublished)MeAsp
Act
ivity
Open questions
• Lattice structure?
• Mechanism of receptor-receptor coupling?
• Do other receptors work this way?
Sto
ck (
20
00)
Conclusions• Signaling properties of the chemotaxis network:
– Precise and robust adaptation– Signal integration– Sensitivity
• FRET studies reveal two regimes of activity– Regime I: low activity and constant KD
– Regime II: high activity and increasing KD
• Model of coupled 2-state receptors account for signaling properties, and for two regimes– Regime I (Δε > 0): coupling → enhanced sensitivity– Regime II (Δε < 0): coupling → enhanced
cooperativity (but only for homogeneous clusters)• Adaptation “homogenizes” receptors (Δε ≈ 0) for
enhanced sensitivity