Transcript Document 7627002

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
Segall, Block, and Berg (1986)
• “Precise and robust
adaptation”: range of 34 orders of magnitude
of attractant
• “Signal integration”:
multiple attractants
CCW vs CW bias for tethered cells
in response to step in attractant
• “Sensitivity”:
amplification
http://www.rowland.harvard.edu/l
abs/bacteria/projects_fret.html
The chemotaxis network
…but signal integration and sensitivity
are still not well understood.
Chemoreceptors
Dimer
Tar - aspartate, glutamate (~900 copies)
Sensor
Tsr - serine (~1600)
Trg - ribose, galactose (~150)
Tap - dipeptides (~150)
(Aer - oxygen via FAD (150?))
Linker region
•Attractant binding inhibits
phosphorylation of CheA
•Adaptation:
More attractant → increased
methylation by CheR → increased
rate of phosphorylation of CheA
Transmembrane
helices
380 A
Methyl binding sites
CheB, CheR
Cytoplasmic
domain
Less attractant → increased
demethylation by CheB → decreased
rate of phosphorylation of CheA
CheA / CheW
binding region
Stock (2000)
Gestwicki et al. (2000)
Chemoreceptor clustering
Receptors are
clustered globally into a
large array, and locally
into trimers of dimers.
Kim et al. (1999); Studdert
and Parkinson (2004)
In vivo FRET studies of receptor
activity
Real-time measurement
of rate of phosphorylation
of CheY.
FRET also allows
subcellular imaging,
Vaknin And Berg (2004).
Sourjik and Berg (2002)
FRET data: two regimes of activity
Sourjik and Berg (2002)
Regime I:
• Activity moderate to low at
zero ambient MeAsp (0.06,1)
• KD small and almost constant
Regime II
Regime I
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
Two regimes of receptor activity
consistent with 2-state receptor model.
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
d Methylatio n
 a[CheR] - b[CheB]  Activity 
dt
– Therefore, at steady state,
 Activity   a [CheR] / b [CheB]
Barkai and
Leibler (1997)
– Which implies precise and robust adaptation of
each receptor complex to a fixed activity.
Two regimes of a 2-state receptor
On
But first, aOff
“1-state” receptor:
Off
Regime I
Regime II
Off
Off
On
Off
Regime I:
• Activity low to very low at
zero ligand concentration
• KD = KDoff
On
Free
Energy
Off
On
Ligand
Regime II:
• Activity high (saturated)
at
1
Pno ligand

bound
zero
ligand
concentration
C
on
1

• KD increasing as ε ↓
KD
KD
Ligand
Ligand
on


e
Activity P
However,
single
receptor
does notoff
on 
on
off
  e  C e
e
account for low apparent KD in
off Regime I.
KD
Receptor-receptor coupling
Duke and Bray (1999)
Duke and Bray (1999)
proposed that receptorreceptor coupling could
enhance sensitivity to ligands.
Toy model: if N receptors are all “on” or all “off” together,
Activity  Pon 
1
1  e N (1 
C
N
)
off
,    on   off
KD
Receptor-receptor coupling gives
enhanced sensitivity (low KD) in
Regime I, and enhanced cooperativity
(high Hill coefficient ) in Regime II.
Regime I (Δε > 0):
• Low activity ~ e-NΔε at
zero ligand concentration
• KD = KDoff / N
• Hill coefficient = 1
Regime II (Δε < 0):
• KD = KDoff e-Δε
• Hill coefficient = N
Review of FRET data
Regime I
• Low, constant KD = KDoff /N, value of N ?
• 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 ?
More
Tars
Less
Tars
Hill coefficient increases with
receptor homogeneity.
Must consider mixture of different
receptor types.
Sourjik and Berg (2004)
Model: 1d mixed lattice of receptors
Normalized Activity
EJ
Tar
Tsr
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
Log([MeAsp])
Receptor homogeneity and
cooperativity
Normalized Activity
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.
Log([MeAsp])
More
Tars
Less
Tars
Adaptation
Adaptation uses methylation to return all receptors
to Δε ≈ 0, and thereby enhances sensitivity.
Δε >
≈0
≈0
Δε >
0
Δε
≈0
Off
Tar
Tsr
Δε ≈
>0
0
Precise adaptation
Scaling of wild-type response data
Sourjik and Berg: Δ[MeAsp]
→ ΔFRET{Tar(QEQE)}
Sourjik and Berg (2002)
“Free energy” scaling:
Δ[MeAsp] → Δ(Fon – Foff)
ΔFRET for Tar(QEQE) strain
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
Activity
•
MeAsp
Sourjik (unpublished)
Open questions
• Lattice structure?
• Mechanism of receptor-receptor coupling?
Stock (2000)
• Do other receptors work this way?
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