The "pH-Activated Trigger" Mechanism of Colicin E1 Channel Domain Abdi Musse MSc. Final Examination Supervisor Dr. A. R. Merrill Advisory committee Dr.

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Presentation transcript:

The "pH-Activated Trigger" Mechanism of Colicin E1 Channel Domain Abdi Musse MSc. Final Examination Supervisor Dr. A. R. Merrill Advisory committee Dr. G. Harauz Dr. F. J. Sharom

Outline 1.Introduction 2.Research Objectives 3.Results and Discussion 4.Summary and Conclusions

Overview 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 + ) Colicin E1

R C T Colicin Ia Wiener et al. (1997) Structure and Function H2NH2N COOH RCT BtuB Receptor Tol Network (TolC, A and Q) Channel-forming

The Channel Domain Elkins et al. (1997) H1 H2 H3 H10 H4 H7 H6 H5 H8 H9 2.5 Å Structure of P190 Three- layered sandwich structure

Interactions with Membranes Activated-intermediate Membrane-anchored Precursor

Formations of the Open Channel   The precursor The open channel Monomer 4 – 9 Å diameter Voltage-gated

Mechanism 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 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) H4 H5a

The 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

Mutant Proteins of Colicin E1 Asp  Ser D410S D408S D408S/D410S D408S/D423S D410S/D423S D408S/D410S/D423S Ala  Cys A407C/A411C Single Trp F413W F413W/D408S/D423S

Cytotoxicity Cytotoxicity

Structural Integrity WT (folded) WT (7 M GnHCl)

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

Presence of a Disulfide Bond in A407C/A411C Channel Peptide MIANS fluorescence WT (GnHCl) A407C/A411C (GnHCl) WT (folded) A407C/A411C (folded) Stoichiometry of MIANS Conjugation

Membrane Binding Membrane Binding TNP Fluorescence Quenching

Typical Binding Profile for the WT Channel Peptide pK a  4

The Expected profile for the Asp  Ser Mutants

b

The Expected Profile for the Disulphide Bonded Mutant b c

The Binding Profile for the WT protein Expected pH-binding profile The effective pK a  4.1 (0.1)

The Binding Profiles of the Double Asp  Ser Mutants Alkaline-directed shift in binding profile Consistent with the predicted profile of an altered trigger mechanism

The Binding Profiles of the Disulphide Bonded Mutant A407C/A411C Un-expected binding profile At pH 4.5: K a = 1.4 (0.2) M -1 (reduced) 1.7 (0.3) M -1 (oxidized)

Membrane Insertion Fluorescence Quenching

Time Course of the Fluorescence Quenching

Apparent Rates of Membrane Insertion D408 H-bond D410 Salt bridge

In vitro Channel Activity Cl - Efflux Cl - Fluorescence Dequenching

Time Course of the Fluorescence Dequenching

The Initial Rate of Cl - Efflux

W-424 W-413

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

Time-resolved and Steady-state Fluorescence Parameters

The Time-resolved and Steady-state Fluorescence Parameters

Time-resolved and Steady-state Fluorescence Parameters

The Trigger Residues

The Topology of the Trigger Motif

Possible Implications for the in vivo Mechanism of Activation H1 Docking site Trigger

Summary 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

Acknowledgements 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