the "ph-activated trigger" mechanism of colicin e1 channel domain abdi musse msc. final...
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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