Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac

The four salt bridges (Asp222-Arg28I, Arg233-Glu288, Arg234-Glu274, and Asp242-Arg265) linking domains I and II in Crv1Aa were abolished individually in x-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.

Bibliographic Details

Main Authors:	Coux, Florence, Vachon, Vincent, Rang, Cécile, Moozar, Kouros, Masson, Luke, Royer, Monique, Bes, Martine, Rivest, Sébastien, Brousseau, Roland, Schwartz, Jean Louis, Laprade, Raynald, Frutos, Roger
Format:	article biblioteca
Language:	eng
Subjects:	H10 - Ravageurs des plantes, Bacillus thuringiensis, toxine bactérienne, biopesticide, lutte anti-insecte, transfert de gène, Manduca sexta, http://aims.fao.org/aos/agrovoc/c_761, http://aims.fao.org/aos/agrovoc/c_9047, http://aims.fao.org/aos/agrovoc/c_27467, http://aims.fao.org/aos/agrovoc/c_3885, http://aims.fao.org/aos/agrovoc/c_24846, http://aims.fao.org/aos/agrovoc/c_30350,
Online Access:	http://agritrop.cirad.fr/488238/ http://agritrop.cirad.fr/488238/1/488238.pdf
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