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J. Biol. Chem., Vol. 275, Issue 31, 23602-23607, August 4, 2000
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From the Department of Biological Chemistry, The Weizmann Institute
of Science, Rehovot 76100, Israel
The pore-forming domain of Bacillus
thuringiensis Cry1Ac insecticidal protein comprises of a seven
Insertion and Organization within Membranes of the
-Endotoxin
Pore-forming Domain, Helix 4-Loop-Helix 5, and Inhibition of Its
Activity by a Mutant Helix 4 Peptide*
-helix bundle (1-7). According to the "umbrella model,"
4 and 5 helices form a hairpin structure thought to be inserted
into the membrane upon binding. Here, we have synthesized and
characterized the hairpin domain, 4-loop-5, its 4 and 5
helices, as well as mutant 4 peptides based on mutations that
increased or decreased toxin toxicity. Membrane permeation studies
revealed that the 4-loop-5 hairpin is extremely active compared
with the isolated helices or their mixtures, indicating the
complementary role of the two helices and the need for the loop for
efficient insertion into membranes. Together with spectrofluorometric
studies, we provide direct evidence for the role of 4-loop-5 as
the membrane-inserted pore-forming hairpin in which 4 and 5 line
the lumen of the channel and 5 also participates in the
oligomerization of the toxin. Strikingly, the addition of the active
4 mutant peptide completely inhibits 4-loop-5 pore formation,
thus providing, to our knowledge, the first example that a mutated
helix within a pore can function as an "immunity protein" by
directly interacting with the segments that form the pore. This
presents a potential means of interfering with the assembly and
function of other membrane proteins as well.
*
This research was supported by the U. S. A.-Israel
Binational Agriculture Research and Development Fund (BARD)
Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 972-8-9342711;
Fax: 972-8-9344112; E-mail: Yechiel.Shai@weizmann.ac.il.
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