Dataset Information

Rearrangement of N-Terminal ?-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

ABSTRACT: Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal ?-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within ?-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix ?-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix ?-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices ?-2b and ?-3 form an extended helix ?-3 necessary for oligomer assembly of Cry toxins.

SUBMITTER: Pacheco S

PROVIDER: S-EPMC7601232 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Rearrangement of N-Terminal α-Helices of <i>Bacillus thuringiensis</i> Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Pacheco Sabino S Quiliche Jean Piere Jesus JPJ Gómez Isabel I Sánchez Jorge J Soberón Mario M Bravo Alejandra A

Toxins 20201008 10

Cry proteins produced by <i>Bacillus thuringiensis</i> are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular re ...[more]

PMID: 33049917

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Project description:The fall armyworm, Spodoptera frugiperda, is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III β-sheets β-16 and β-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III β-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor.IMPORTANCESpodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III β-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.

Dataset Information

Rearrangement of N-Terminal ?-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Publications

Rearrangement of N-Terminal α-Helices of <i>Bacillus thuringiensis</i> Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Similar Datasets

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