1. Modeling the chemosensing system of E. coli
Ned Wingreen – Princeton
Juan Keymer – Princeton
Robert Endres – Princeton/NEC
Yigal Meir – Ben Gurion University

2. 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?

3. 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)
J. E. Segall, S. M. Block, and H. C. Berg, Proc Natl Acad Sci U S A Dec;83(23): – Fig. 2
CCW vs CW bias for tethered cells in response to step in attractant

4. The chemotaxis network
…but signal integration and sensitivity are still not well understood.

5. Chemoreceptors Dimer Tar - aspartate, glutamate (~900 copies)
Tsr - serine (~1600)
Trg - ribose, galactose (~150)
Tap - dipeptides (~150)
(Aer - oxygen via FAD (150?))
Sensor
Transmembrane helices
Linker region
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
380 A
Methyl binding sites
CheB, CheR
Cytoplasmic
domain
CheA / CheW
binding region
Stock (2000)

6. Chemoreceptor clustering
Receptors are clustered globally into a large array, and locally into trimers of dimers.
Gestwicki et al. (2000)
Gestwicki et al. J. Bacteriol. 182, (2000) – Immunofluorescence images using anti-Trg antibody.
Kim et al. (1999); Studdert and Parkinson (2004)

7. 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).
IMPORTANT ADVANTAGE OF FRET IS THAT CHEY-P IS UPSTREAM OF MOTOR.
A. Vaknin and H. C. Berg, Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system, Proc Natl Acad Sci U S A Dec 7;101(49):
Sourjik and Berg (2002)

8. FRET data: two regimes of activity
Sourjik and Berg (2002)
Regime I
Regime II
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 ( )
KD1 large and increasing with methylation
Plateau in activity
KD2 approximately constant
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Two regimes of receptor activity consistent with 2-state receptor model.

9. 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.
Barkai and Leibler (1997)

10. Two regimes of a 2-state receptorOn
Off
But first, a “1-state” receptor:
Regime I
Regime II
Regime I:
Activity low to very low at zero ligand concentration
KD = KDoff
Regime II:
Activity high (saturated) at zero ligand concentration
KD increasing as εon ↓
Off
Off On On
Free Energy
Off
Off
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7. On KD
Ligand
Ligand
Ligand
However, single receptor does not account for low apparent KD in Regime I.

11. Receptor-receptor coupling
Duke 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,
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
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Receptor-receptor coupling gives enhanced sensitivity (low KD) in Regime I, and enhanced cooperativity (high Hill coefficient ) in Regime II.

12. Review of FRET data 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 ?
= KDoff /N, value of N ?
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
More Tars
Less Tars
Hill coefficient increases with receptor homogeneity.
Must consider mixture of different receptor types.
Sourjik and Berg (2004)

13. Model: 1d mixed lattice of receptors
Tsr
Tar EJ
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
Normalized Activity
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Log([MeAsp])

14. Receptor homogeneity and cooperativity
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.
Normalized Activity
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Log([MeAsp])
More Tars
Less Tars

15. Adaptation
Adaptation uses methylation to return all receptors to Δε ≈ 0, and thereby enhances sensitivity.
Tsr
Tar
Δε ≈ 0
Δε > 0
Δε ≈ 0
Δε > 0
Δε ≈ 0
Δε > 0
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Off
Δε ≈ 0

16. Precise adaptation
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.

17. Scaling of wild-type response data
Sourjik and Berg: Δ[MeAsp] → ΔFRET{Tar(QEQE)}
“Free energy” scaling: Δ[MeAsp] → Δ(Fon – Foff)
ΔFRET for Tar(QEQE) strain
Sourjik and Berg (2002)
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Doesn’t collapse zero-ambient data.
Includes zero-ambient data!

18. Predictions For homogeneous lattice –
Transition from Regime I to Regime II with methylation
Adaptation range set by KDon
Activity
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
MeAsp
Sourjik (unpublished)

19. Open questions Lattice structure?
Mechanism of receptor-receptor coupling?
Do other receptors work this way?
C. A. Hale, H. Meinhardt, and P. A. J. de Boer, EMBO J. 20, (2001) – Fig. 7.
Stock (2000)

20. 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