The role of a proline‐induced broken‐helix motif in α‐helix 2 of Bacillus thuringiensis δ‐endotoxins

Abstract

Bacillus thuringiensis N N-endotoxins (Cry proteins), are widely used for insect control and plant protection. They are water-soluble proteins that insert into membranes forming ion channels. In most Cry toxins K K-helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken-helix motif. The flexibility of the motif was altered through site-directed mutagenesis. It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein. By removing the broken-helix motif, the biological properties were restored to a wild type level. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

“…These studies did not address the fate of regions of the toxin other than the ␣-helices of domain I once the toxin is inserted. However, proteinase K protection studies (typically used for detecting the regions of membrane proteins inside lipid bilayer) have shown that a 60-kDa form (or higher molecular weight aggregate) of the toxin has been protected in membranes (15,18,19,31,35). This suggests that most of the toxin was likely to be embedded in the membrane.…”

“…The partitioning rate of every mutant into the brush border membrane and artificial membranes are Entire Crystal Toxin Inserts into BBMV not the same. We have seen that mutagenesis of Phe-371 to other residues in decreasing order of hydrophobicity have compromised its insertion ability (31,34). Although mutation to Trp retained most of the toxicity of the toxin in these studies, the difference in the amount protected for this mutant (Fig.…”

“…In addition the time lag (T 0 ) values of 100 ng of all mutants ranged from 4.5 to 6.5 min for all the mutants while that of same amount of Cry1Aa and Cry1Ab was 5.0 min. This time lag is an indicator of the time the toxin takes, after adding to the membrane, to initiate pore formation, and in other words, it is the time of partitioning of the toxin into BBMV (28,31,32) suggesting that all our mutants partitioned into the membrane at a similar rate. The exception to this is Phe-371 when mutated to Cys or Ala, as reported in our earlier studies (34, 35), which is restored to almost wild type with tryptophan substitution.…”

Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity

“…However, we cannot rule out the possibility that buried disulfide bridges may not be exposed to the reducing environment of the midgut lumen. In this case, a plausible explanation is that the entire domain I inserts into membrane as previously proposed, either as a whole protein [7,[12][13][14][15][16][17] or as a member of an oligomeric complex [18,23,35,36]. This would also result in no difference in toxicity between the wild-type and disulfide proteins.…”

“…Both models involve separation of the α-helices from the α-helical bundle, followed by their insertion into the target membranes resulting in the formation of ion pores. Other studies have proposed that the whole domain I inserts into the protein, without α-helical separation [7,[12][13][14][15][16][17].…”