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Applied and Environmental Microbiology, January 2009, p. 359-365, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01930-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Helix {alpha}4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation{triangledown}

Frédéric Girard,1 Vincent Vachon,1 Gabrielle Préfontaine,2 Lucie Marceau,1 Jean-Louis Schwartz,1 Luke Masson,2 and Raynald Laprade1*

Groupe d'Étude des Protéines Membranaires, Université de Montréal,1 Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada2

Received 19 August 2008/ Accepted 11 November 2008

Helix {alpha}4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix {alpha}4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix {alpha}4 is involved mainly in the postbinding steps of pore formation.

* Corresponding author. Mailing address: Groupe d'Étude des Protéines Membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec H3C 3J7, Canada. Phone: (514) 343-7924. Fax: (514) 343-7146. E-mail: raynald.laprade{at}umontreal.ca

{triangledown} Published ahead of print on 14 November 2008.

Applied and Environmental Microbiology, January 2009, p. 359-365, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01930-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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