the "ph-a c tivated trigger" mechanism of colicin e1 channel domain

The "pH-AThe "pH-Acctivated Trigger" Mechanism tivated Trigger" Mechanism of Colicin E1 Channel Domainof Colicin E1 Channel Domain

Abdi Musse

MSc. Final Examination

Supervisor

Dr. A. R. Merrill

Advisory committee

Dr. G. Harauz

Dr. F. J. Sharom

OutlineOutline

1. Introduction

2. Research Objectives

3. Results and Discussion

4. Summary and Conclusions

OverviewOverview

The Biology of Pore-forming Colicins

• Antimicrobial proteins that are secreted by Escherichia coli

• Targets the cytoplasmic membrane

• Forms lethaly depolarizing ion channels

• Dissipations of the cationic gradients (H+, K+, Na+)

A T PA D P + P i

N a + , H +

K + N a + , H +

A T PA D P + P i

N a + , H +

N a + , H +H +

A T PA D P + P i

N a + , H +

K + N a + , H +

A T PA D P + P i

N a + , H +

N a + , H +H +

Colicin E1

Colicin Ia

Wiener et al. (1997)

Structure and FunctionStructure and Function

H2N COOHR CT

BtuB Receptor

Tol Network(TolC, A and Q)

Channel-forming

The Channel DomainThe Channel Domain

Elkins et al. (1997)

H5H8H9

• 2.5 Å Structure of P190

• Three- layered sandwich structure

Interactions with MembranesInteractions with Membranes

Activated-intermediate

Membrane-anchored Precursor

Formations of the Open ChannelFormations of the Open Channel

The precursor

The open channel

• Monomer

• 4 – 9 Å diameter

Voltage-gated

Mechanism of ActivationMechanism of Activation

• Acid-induced activation is common to most toxins

• Onset of Protein unfolding

• Increased structural flexibility

• Potentiates the massive unfloding events requisite for

membrane insertion and channel formation

The pH-Activated Trigger HypothesisThe pH-Activated Trigger Hypothesis

• The trigger motif: helices 4 and 5a

• Activating helix-to-coil transition of the trigger motif

• Disruption of the critical H-bonds formed by D-408, D-410

and D-423

Merrill et al. (1997)

The Research ObjectivesThe Research Objectives

Purpose

• To test the proposed pH-activated trigger mechanism

Approaches

1. Replacements of the critical acidic residues with serine

2. Incorporation of a disulphide bond within the trigger motif

• Membrane binding

• Insertion kinetics

• Channel activity

• Structural elucidations

Mutant Proteins of Colicin E1Mutant Proteins of Colicin E1

A411CA407C

Asp Ser

• D410S

• D408S/D410S D408S/D423S

D410S/D423S

• D408S/D410S/D423S

Ala Cys

• A407C/A411C

Single Trp

• F413W

• F413W/D408S/D423S

CytotoxicityCytotoxicity

Structural IntegrityStructural Integrity

P r o t e i n

maxλ em( n m )

W T 3 1 9

D 4 0 8 S 3 2 2

D 4 1 0 S 3 2 2

D 4 0 8 S / D 4 2 3 S 3 1 9

D 4 0 8 S / D 4 1 0 S 3 2 2

D 4 1 0 S / D 4 2 3 S 3 2 0

A 4 0 7 C / A 4 1 1 C 3 2 0

W T ( 7 M G n H C l )

tive F

WT (folded)

WT (7 M GnHCl)

Probing Free Sulfahydral Side-chains in Probing Free Sulfahydral Side-chains in A407C/A411C with MIANSA407C/A411C with MIANS

OProtein-SH

Protein-S

Non-fluorescent

Presence of a Disulfide Bond in A407C/A411C Presence of a Disulfide Bond in A407C/A411C Channel PeptideChannel Peptide

350 400 450 500 550 600

tive F

MIANS fluorescence

A407C A411C

WT (GnHCl)

A407C/A411C (GnHCl)

WT (folded)

A407C/A411C (folded)

Stoichiometry of MIANS Conjugation

Membrane BindingMembrane Binding

TNPTNP TNPTNP

Fluorescence Quenching

Typical Binding Profile for the WT Channel Typical Binding Profile for the WT Channel PeptidePeptide

3.5 4.0 4.5 5.0

The Expected profile for the Asp The Expected profile for the Asp Ser Ser MutantsMutants

3.5 4.0 4.5 5.0

Membrane Binding

3.5 4.0 4.5 5.0

The Expected profile for the Asp The Expected profile for the Asp Ser Ser Mutants Mutants

bD423S

A411CA407C

The Expected Profile for the Disulphide The Expected Profile for the Disulphide Bonded MutantBonded Mutant

3.5 4.0 4.5 5.0

The Binding Profile for the WT proteinThe Binding Profile for the WT protein

• Expected pH-binding profile

• The effective pKa 4.1 (0.1)

3.5 4.0 4.5 5.0

WT D408S/ D423S WT D408S/ D423S

The Binding Profiles of the Double AspThe Binding Profiles of the Double AspSer Ser MutantsMutants

3.5 4.0 4.5 5.0

WT D410S/ D423S WT D410S/ D423S

3.5 4.0 4.5 5.0

WT D408S/ D423S WT D408S/ D423S

3.5 4.0 4.5 5.0

WT D408S/ D410S WT D408S/ D410S

WT D408S/ D423S WT D408S/ D423S

• Alkaline-directed shift in binding profile

• Consistent with the predicted profile of an altered

trigger mechanism

The Binding Profiles of the Disulphide Bonded The Binding Profiles of the Disulphide Bonded MutantMutant

3.5 4.0 4.5 5.0

A407C/A411C

WT D408S/ D423S WT D408S/ D423S

• Un-expected binding profile

• At pH 4.5: Ka = 1.4 (0.2) M-1 (reduced)

1.7 (0.3) M-1

(oxidized)

Membrane InsertionMembrane Insertion

Fluorescence Quenching

Time Course of the Fluorescence QuenchingTime Course of the Fluorescence Quenching

tive F

Time (s)

0 100 200 300 400 5000.3

WT D408SD410SD408S/ D423SD410S/ D423SA407C/ A411C

Apparent Rates of Membrane Insertion Apparent Rates of Membrane Insertion

H-bond

Salt bridge

0Protein 1ms

WT 2.02 (0.10) 1.0D408S 2.44 (0.48) 1.2

D410S 3.91 (0.98) 1.9

A407C/A411C 4.39 (0.29) 2.2*A407C/A411C 5.43 (0.65) 2.5

D408S/D423S 8.88 (1.28) 4.4

D410S/D423S 10.14 (1.81) 5.0

0Protein 1ms

WT 2.02 (0.10) 1.0D408S 2.44 (0.48) 1.2

D410S 3.91 (0.98) 1.9

A407C/A411C 4.39 (0.29) 2.2*A407C/A411C 5.43 (0.65) 2.5

D408S/D423S 8.88 (1.28) 4.4

D410S/D423S 10.14 (1.81) 5.0

In vitroIn vitro Channel Activity Channel Activity

Cl-Cl-

Efflux

SPQSPQ

Cl- Cl-

Cl-Cl-

SPQSPQ

Cl- Cl-

Cl-Cl-

Fluorescence Dequenching

0 50 100 150 200 250 300

pH 5.0

0 100 200 300 400 500 600

pH 6.0

Time Course of the Fluorescence Time Course of the Fluorescence DequenchingDequenching

Time (s)

tive F

Time (s)

WT D408SD410SD408S/ D423SD410S/ D423SA407C/ A411C

The Initial Rate of ClThe Initial Rate of Cl-- Efflux Efflux

0v(x102 % Fmaxs-1)

Rel.'0v (x102% Fmax s

Rel.'0v

Protein pH 5.0 pH 6.0

WT 14.9 (0.1) 1.0 0.30 (0.03) 1.0

D408S 51.1 (0.2) 3.4 2.31 (0.04) 7.7

D410S 63.0 (0.2) 4.2 3.72 (0.03) 12.4

A407C/A411C 216 (3) 14.5 21.1 (0.1) 70.3*A407C/A411C 86.2 (1.7) 5.8 11.1 (0.3) 37.0

D408S/D423S 117 (2) 7.9 10.7 (0.1) 35.7

D410S/D423S 159 (1) 10.7 11.7 (0.3) 30.7

0v(x102 % Fmaxs-1)

Rel.'0v (x102% Fmax s

Rel.'0v

Protein pH 5.0 pH 6.0

WT 14.9 (0.1) 1.0 0.30 (0.03) 1.0

D408S 51.1 (0.2) 3.4 2.31 (0.04) 7.7

D410S 63.0 (0.2) 4.2 3.72 (0.03) 12.4

A407C/A411C 216 (3) 14.5 21.1 (0.1) 70.3*A407C/A411C 86.2 (1.7) 5.8 11.1 (0.3) 37.0

D408S/D423S 117 (2) 7.9 10.7 (0.1) 35.7

D410S/D423S 159 (1) 10.7 11.7 (0.3) 30.7

The Time-resolved and Steady-state The Time-resolved and Steady-state Fluorescence of the Single Trp MutantsFluorescence of the Single Trp Mutants

pH 1 2 c1 c2 SVR QF maxSSPeptide(ns) (ns) (nm)

F413W 3.5 4.41 1.03 0.62 0.38 1.16 1.93 0.19 (0.01) 323 (1)

F413W 6.0 3.94 1.26 0.48 0.52 1.09 2.03 0.12 (0.00) 325 (2)

F413W/D408S/D423S 3.5 4.96 1.15 0.72 0.28 1.11 1.90 0.19 (0.01) 325 (2)