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J. Biol. Chem., Vol. 278, Issue 27, 24491-24499, July 4, 2003

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The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1^*,

Abdiwahab A. Musse and A. Rod Merrill 

From the Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada

The in vitro activity of the channel-forming bacteriocins such as colicin E1 in model membranes requires the specific activation of the protein by an acidic environment in the presence of a membrane potential. Acid activation of the C-terminal domain results in the formation of an insertion-competent intermediate with an enhanced ability to penetrate and perforate cell membranes. We report novel findings of this activation process through the design and study of mutant proteins involving the replacement of conserved Asp residues Asp-408, Asp-410, and Asp-423 within helices 5_a and 4 in the colicin E1 channel domain that resulted in enhanced membrane binding, bilayer insertion rates, and ion channel activities at near neutral pH values. This activation process involves the destabilization of a critical salt bridge (Asp-410 and Lys-406) and H-bonds (Asp-408 and Ser-405 main chain; Asp-423 and Lys-420 main chain). The helix-to-coil transition of this motif was identified previously by time-resolved Trp fluorescence measurements (Merrill, A. R., Steer, B. A., Prentice, G. A., Weller, M. J., and Szabo, A. G. (1997) Biochemistry 36, 6874–6884), and here we use this approach to demonstrate that disruption of the helical structure of helices 4 and 5_a results in a shift in this equilibrium to favor the coil state. Finally, we show that the essential components of the pH trigger motif are conserved among the channel-forming colicins and that it likely exists within other bacterial proteins and may even have evolved into more sophisticated devices in a number of microbial species.

Received for publication, March 7, 2003 , and in revised form, April 15, 2003.

^* This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional text, Table S1, and Fig. S1.

To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 3806); Fax: 519-766-1499; E-mail: merrill{at}chembio.uoguelph.ca.

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