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J. Biol. Chem., Vol. 276, Issue 38, 35546-35551, September 21, 2001

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Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac^*

Florence Coux^§¶, Vincent Vachon, Cécile Rang^§, Kouros Moozar, Luke Masson, Monique Royer^§, Martine Bes^§, Sébastien Rivest, Roland Brousseau, Jean-Louis Schwartz, Raynald Laprade^**, and Roger Frutos^§

From the  Groupe de recherche en transport membranaire, Université de Montréal, Montreal, Quebec, H3C 3J7, Canada, ^§ IGEPAM, CIRAD, Montpellier, France and the  Biotechnology Research Institute, National Research Council, Montreal, Quebec, H4P 2R2, Canada

The four salt bridges (Asp²²²-Arg²⁸¹, Arg²³³-Glu²⁸⁸, Arg²³⁴-Glu²⁷⁴, and Asp²⁴²-Arg²⁶⁵) linking domains I and II in Cry1Aa were abolished individually in -helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp²⁴²-Arg²⁶⁵ bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

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