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

H +

K +

K +

K +

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

H +

K +

K +

K +

N a + , H +

N a + , H +H +

Colicin E1

R

CT

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)

H1

H2

H3

H10

H4

H7H6

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)

D-423

D-408

D-410

H4

H5a

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

Tools

• Membrane binding

• Insertion kinetics

• Channel activity

• Structural elucidations

D423

D410

D408

D423

D410

D408

Mutant Proteins of Colicin E1Mutant Proteins of Colicin E1

A411CA407C

S S

Asp Ser

• D410S

D408S

• 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 )

3 5 3

(nm)

Rela

tive F

luore

scen

ce (

au

)

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

NH

SO3-N

O

OProtein-SH

NH

SO3-N

O

O

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

0.0

0.2

0.4

0.6

0.8

1.0

(nm)

Rela

tive F

luore

scen

ce (

au

)

MIANS fluorescence

A407C A411C

C-505

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

pKa 4

pH

Mem

bra

ne B

ind

ing

D423

D410

D408

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

3.5 4.0 4.5 5.0

D423S

D410S

D408S

pH

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

D410S

D408S

A411CA407C

S S

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

c

3.5 4.0 4.5 5.0

b

c

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

0.1

1

10

pH

Ka(x

10

6 M

-1)

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

0.1

1

10

WT D410S/ D423S WT D410S/ D423S

pH pH

Ka(x

10

6 M

-1)

3.5 4.0 4.5 5.0

0.1

1

10

WT D408S/ D423S WT D408S/ D423S

3.5 4.0 4.5 5.0

0.1

1

10

pH

WT D408S/ D410S WT D408S/ D410S

WT D408S/ D423S WT D408S/ D423S

WT D408S/ D423S WT D408S/ D423S

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

0.1

1

10

Ka(x

10

6 M

-1)

pH

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

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Br

Time Course of the Fluorescence QuenchingTime Course of the Fluorescence Quenching

Rela

tive F

luore

scen

ce (

au

)

Time (s)

0 100 200 300 400 5000.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

WT D408SD410SD408S/ D423SD410S/ D423SA407C/ A411C

Apparent Rates of Membrane Insertion Apparent Rates of Membrane Insertion

D408

H-bond

D410

Salt bridge

kk '

0Protein 1ms

Rel.

'0

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

kk '

0Protein 1ms

Rel.

'0

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

SPQ

SPQSPQ

SPQ

SPQ

SPQ

SPQ

SPQSPQ

Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

SPQ

SPQSPQ

SPQ

SPQ

SPQ

SPQ

SPQSPQ

Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Fluorescence Dequenching

0 50 100 150 200 250 300

0

10

20

30

40

50

60

pH 5.0

0 100 200 300 400 500 600

0

10

20

30

40

50

pH 6.0

Time Course of the Fluorescence Time Course of the Fluorescence DequenchingDequenching

Time (s)

Rela

tive F

luore

scen

ce (

%F

max)

Time (s)

WT D408SD410SD408S/ D423SD410S/ D423SA407C/ A411C

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

'0v '

0v(x102 % Fmaxs-1)

Rel.'0v (x102% Fmax s

-1)

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 '

0v(x102 % Fmaxs-1)

Rel.'0v (x102% Fmax s

-1)

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

W-424

W-413

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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

2pH 11 22 c1c1 c2c2 SVR QF maxSS 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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

22

Time-resolved and Steady-state Time-resolved and Steady-state Fluorescence ParametersFluorescence Parameters

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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

2pH 11 22 c1c1 c2c2 SVR QF maxSS 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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

22

The Time-resolved and Steady-state The Time-resolved and Steady-state Fluorescence ParametersFluorescence Parameters

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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

2pH 11 22 c1c1 c2c2 SVR QF maxSS 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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

22

The Time-resolved and Steady-state The Time-resolved and Steady-state Fluorescence ParametersFluorescence Parameters

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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

2pH 11 22 c1c1 c2c2 SVR QF maxSS 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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

22

Time-resolved and Steady-state Time-resolved and Steady-state Fluorescence ParametersFluorescence Parameters

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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

2pH 11 22 c1c1 c2c2 SVR QF maxSS 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)

F413W/D408S/D423S 6.0 4.63 1.29 0.55 0.45 1.09 1.99 0.15 (0.01) 326 (3)

22

10 20 30 40 50 60 | | | | | |E1 392 AFEKYKDVLNKKFSKADRDAIFNALASVKYDDWAKHLDQFAKYLKITGHVSFGYDVVSDI5 360 AFEKYKNVLDKKISKVDRDAIFNALESVNYDELSKNLTKISKSLKITSRVSFLYDVGSDFK 417 AFEKYKNVLDKKFSKVDRDAIFNALESVNYDELSKNLTKISKSLKITSRVSFLYDVGSDF10 360 AFEKYKNVLDKKFSKVDRDDIFNALESITYDEWAKHLEKISRALKVTGYLSFGYDVWDGTA 450 SLNKITANPAMKINKADRDALVNAWKHVDAQDMANKLGNLSKAFKVADVVMKVEKVREKSN 246 SLNKVLANPKMKVNKSDKDAIVNAWKQVNAKDMANKIGNLGKAFKVADLAIKVEKIREKSB 371 SINKLMANPSLKINATDKEAIVNAWKAFNAEDMGNKFAALGKTFKAADYAIKANNIREKSU 477 SVNKLMANPDLKINAADRDAIVNAWKAFDAEDMGNKFAALGKTFKAADYVMKANNVREKSY 486 SVNKLMANPDLKINAADRDVIVNAWKAFDAEDMGNKFAALGKTFKAADYVMKANNVREKSIa 496 TYEKYRADINKKINAKDRAAIAAALESVKLSDISSNLNRFSRGLGYAGKFTSLADWITEFIb 496 AFDKFRNNLNKKYNIQDRMAISKALEAINQVHMAENFKLFSKAFGFTGKVIERYDVAVEL : :* * . *: : * . . ..:: :.: : :. .

The Trigger Residues The Trigger Residues

The Topology of the Trigger Motif The Topology of the Trigger Motif

Colicin E1

Colicin I a Colicin N

Colicin A

Possible Implications for the Possible Implications for the in vivoin vivo Mechanism of Activation Mechanism of Activation

H1

H1

Docking site

Trigger

Preplasm

I nner Membrane

Outer Membrane

Summary and ConclusionsSummary and Conclusions

• These observations confirm the proposed pH-activated trigger mechanism of colicin E1

• Asp Ser mutations disrupted criticall H-bonds within

the tirgger motif

• Elevated binding, insertion, and channel activities at

near-neutral pH

• Shift in the helix-to-coil transition of the trigger motif

toward random Coil-like conformational state for helix 4

AcknowledgementsAcknowledgements

Advisor Dr. A. R. Merrill

Examining Committee

Dr. G. Harauz

Dr. P. D. Josephy

Dr. M. Palmer

Colleagues in the Merrill Laboratory

Tanya Brodeur

Susan Yates

Tania Roberts

Gerry Prentice*

Dave Teal

Zahir Hussein

*Special thanks

Advisory Committee

Dr. G. Harauz

Dr. F. J. Sharom