Location of the <i>Bombyx mori</i> 175 kDa cadherin‐like protein‐binding site on <i>Bacillus thuringiensis</i> Cry1Aa toxin

Atsumi, Shogo; Inoue, Yukino; Ishizaka, Takahisa; Mizuno, Eri; Yoshizawa, Yasutaka; Kitami, Madoka; Satō, Ryōichi

doi:10.1111/j.1742-4658.2008.06634.x

Cited by 30 publications

(30 citation statements)

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“…Regarding the interaction of Cry1Ab monomer with Bt-R 1 receptor, previous work showed that domain II, loop ␣-8, loop 2, and loop 3 are involved in the binding of Cry1Ab toxin to this receptor (2,16,32 (17,18). Monomeric structures of the three loop 3 mutants characterized here were only affected in the binding with the CR12 fragment of Bt-R 1 and not with a Bt-R 1 fragment corresponding to CR7-CR12 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Insect cadherins are composed of an ectodomain formed by 11 or 12 cadherin repeats (CRs), a transmembrane domain, and an intracellular domain (14). The exposed loops of Cry1A domain II have been directly involved in binding with cadherin in M. sexta, H. virescens, and B. mori (15)(16)(17)(18). Three Cry1Ab binding sites were mapped in CR7, CR11, and CR12 of the M. sexta Bt-R 1 (15,19).…”

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confidence: 99%

“…The interaction between Cry1Ab/ Cry1Ac toxins and H. virescens cadherin was analyzed, showing that loop 3 of domain II binds to CR12 of this receptor (17). Accordingly, loop 3 of Cry1Aa binds the corresponding site of B. mori cadherin (Bt-R 175 ) (18). Cry1Ab loop ␣-8 and loop 2 bind with M. sexta Bt-R 1 CR7 and CR11 epitopes (15).…”

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Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

125

135

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Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

125

135

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“…For example, residue Tyr445 in loop 3 of Cry1Aa domain II was identified as being important in binding to the cadherin BtR175 of Bombyx mori (Atsumi et al, 2005) while Val582 in domain III was shown to be important for interaction with aminopeptidase N (Atsumi et al, 2008). In parallel, there have been studies to map regions of receptors that may interact with the toxins (Pigott and Ellar, 2007) but even when a common class of receptor (eg a cadherin) is considered, there is little correspondence between regions interacting with different toxins.…”

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Structural classification of insecticidal proteins – Towards an in silico characterisation of novel toxins

Berry

Crickmore

2017

Journal of Invertebrate Pathology

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Article (Accepted Version) http://sro.sussex.ac.uk Berry, Colin and Crickmore, Neil (2017) Structural classification of insecticidal proteins -towards an in silico characterization of novel toxins. Journal of Invertebrate Pathology, 142. pp. 16-22. ISSN 0022-2011 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/62164/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the URL above for details on accessing the published version. Copyright and reuse:Sussex Research Online is a digital repository of the research output of the University.Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available.Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Accepted Manuscript Abstract:The increasing rate of discovery of new toxins with potential for the control of invertebrate pests through next generation sequencing, presents challenges for the identification of the best candidates for further development. A consideration of structural similarities between the different toxins suggest that they may be functionally less diverse than their low sequence similarities might predict. This is encouraging from the prospective of being able to use computational tools to predict toxin targets from their sequences, however more structure/function data are still required to reliably inform such predictions.Introduction:

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“…It has been proposed that a series of different binding events participate in Cry toxin action, such as the primary binding interaction of activated Cry1A toxin with aminopeptidase N (APN) or alkaline phosphatase (ALP) by domain II loop 3 and ␤-16 of domain III (13)(14)(15). Binding to cadherin-like protein (CAD) follows by means of domain II-exposed loops ␣-8, 2, and 3, promoting cleavage of helix ␣-1 of domain I and inducing toxin oligomerization (14)(15)(16)(17)(18)(19). Finally, it was reported that binding of the oligomeric Cry1A toxin to ALP and APN receptors by means of domain II loop 2 is involved in toxin insertion into membrane and cell lysis (2,15,20).…”

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Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

Zhou

Liu

Liang

et al. 2017

Appl Environ Microbiol

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Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

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Location of the Bombyx mori 175 kDa cadherin‐like protein‐binding site on Bacillus thuringiensis Cry1Aa toxin

Cited by 30 publications

References 54 publications

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Structural classification of insecticidal proteins – Towards an in silico characterisation of novel toxins

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

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