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

From National Research Council Canada

DOI	Resolve DOI: https://doi.org/10.1074/jbc.M101887200
Author	Search for: Coux, Florence; Search for: Vachon, Vincent; Search for: Rang, Cécile; Search for: Moozar, Kouros¹; Search for: Masson, Luke¹; Search for: Royer, Monique; Search for: Bes, Martine; Search for: Rivest, Sébastien; Search for: Brousseau, Roland¹; Search for: Schwartz, Jean-Louis¹; Search for: Laprade, Raynald; Search for: Frutos, Roger
Name affiliation	National Research Council of Canada. NRC Biotechnology Research Institute
Format	Text, Article
Journal title	The Journal of Biological Chemistry
ISSN	0021-9258 1083-351X
Volume	276
Issue	38
Pages	35546–35551
Abstract	The four salt bridges (Asp222–Arg281, Arg233–Glu288, Arg234–Glu274, and Asp242–Arg265) linking domains I and II in Cry1Aa were abolished individually in α-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.
Publication date	2001
Publisher	American Society for Biochemistry and Molecular Biology
Peer reviewed	Yes
NRC number	NRC-BRI-44624
NPARC number	3539034
Export citation	Export as RIS
Report a correction	Report a correction
Record identifier	f8b55028-3dd3-4b42-ab6c-8496641bd130
Record created	2009-03-01
Record modified	2020-03-27

Report a problem on this page

Date modified:: 2020-09-29