Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis

Banyuls, Núria; Quan, Yudong; González-Martínez, Rosa M.; Hernández‐Martínez, Patricia; Ferré, Juan

doi:10.1016/j.jip.2020.107439

Cited by 7 publications

(5 citation statements)

References 49 publications

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“…Their absence in Vip3Aa favoured the protoxin conformation and abrogated toxicity. This is in agreement with previous reports showing a drastic lack of toxicity of C-t truncated Vip3Aa in bioassays with several lepidopteran pests, including S. exigua (Banyuls et al, 2018(Banyuls et al, , 2021Gayen et al, 2012Gayen et al, , 2015Li et al, 2007;Quan & Ferré, 2019;Selvapandiyan et al, 2001). The fact that the protoxin conformation was favoured in the absence of Domains IV and V points out a role for the C-t during remodelling, most likely through the interaction with the epithelial membrane of the cells.…”

Section: Discussionsupporting

confidence: 93%

“…Thus, we can envision that helix α1 may be holding Domain I in the protoxin conformation through interactions with Domain II until the protein has been proteolytically processed in the surroundings of the membrane of the midgut cells, to favour remodelling and interaction with the membrane at the appropriate location and timing. The mutant Vip3Aa_M34L, which carries a point modification inside helix α1, shows enhanced toxicity compared to the WT protein, in agreement with previous results obtained with the Vip3Af protein and the related species Spodoptera littoralis (Banyuls et al, 2021 ). The fact that a point mutation inside this region increases the toxicity can be related to the amphipathic nature of helix α1, increasing its hydrophobicity to improve the interaction with the hydrophobic nature of the membrane since the hydrophobic index of Leu (0.943) is higher than Met (0.738) (Black & Mould, 1991 ).…”

Section: Discussionsupporting

confidence: 91%

“…brush border membrane vesicles (BBMV) and Spodoptera frugiperda Sf21 cultured cells, or for exerting toxicity against the latter (Jiang et al, 2020 ; Quan et al, 2021 ). Nevertheless, many reports have shown that they are necessary for the toxicity of Vip3A proteins in vivo (Banyuls et al, 2018 , 2021 ; Gayen et al, 2012 , 2015 ; Li et al, 2007 ; Quan & Ferré, 2019 ; Selvapandiyan et al, 2001 ).…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Lázaro-Berenguer

Paredes-Martínez

Bel

et al. 2022

Microbial Biotechnology

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Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Lázaro-Berenguer

Paredes-Martínez

Bel

et al. 2022

Microbial Biotechnology

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“…The increase in proteolytic cleavage sites between domain I and domain II resulted in the generation of a mutant (Vip3Aa SS193RA/197RA ) with enhanced insecticidal activity against S. frugiperda [ 13 ]. Replacement of M34 in domain I with leucine increased the insecticidal activity against S. exigua and S. littoralis [ 12 , 26 ]. Mutants in domains IV and V showed improved structural stability and affinity for BBMVs and the insecticidal activity of these mutants against S. frugiperda and Helicoverpa armigera was also significantly improved [ 27 ].…”

Section: Discussionmentioning

confidence: 99%

Domain III β4–β5 Loop and β14–β15 Loop of Bacillus thuringiensis Vip3Aa Are Involved in Receptor Binding and Toxicity

Hou,

Li,

Mao

et al. 2024

Toxins

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Vip3Aa, secreted by Bacillus thuringiensis, is effective at controlling major agricultural pests such as Spodoptera frugiperda. However, to control Vip3Aa resistance evolved in the field by different lepidoptera species, an in–depth study of sequence––structure––activity relationships is necessary to design new Vip3Aa variants. In this study, the four specific loops (β4–β5 loop, β9–β10 loop, β12–β13 loop, and β14–β15 loop) in domain III were selected and four loop mutants were constructed by replacing all residues in each specific loop with alanine. We obtained soluble proteins for three of the loop mutants, excluding the β9–β10 loop. These loop mutants have been characterized by toxicity bioassays against S. frugiperda, proteolytic processing, and receptor binding. These results indicate that the β4–β5 loop and β14–β15 loop are involved in receptor binding and Vip3Aa toxicity. Based on this, we constructed numerous mutants and obtained three single mutants (Vip3Aa–S366T, Vip3Aa–S366L, and Vip3Aa–R501A) that exhibited significantly increased toxicity of 2.61–fold, 3.39–fold, and 2.51–fold, respectively. Compared to Vip3Aa, the receptor affinity of Vip3Aa–S366T and Vip3Aa–S366L was significantly enhanced. Furthermore, we also analyzed and aligned the three–dimensional structures of the mutants and Vip3Aa. In summary, these results indicate that the loops in domain III have the potential to be targeted to enhance the insecticidal toxicity of the Vip3Aa protein.

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“…The mutation E168A was selected based on previous studies showing that Vip3Af E168A loses toxicity but retains the tetrameric structure and we hypothesized that could also behave as a DIP mutant 14 , 16 . The DIP proteins Vip3Aa S164C L166C, Vip3Aa E168A and Vip3Ca S164C L166C were generated for this work by SDM following the protocol described elsewhere 37 , using the WT clones as templates. Primers and annealing temperatures used for SDM Polymerase Chain Reaction (PCR) are collected in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

Lázaro-Berenguer

Quan

Hernández‐Martínez

et al. 2022

Sci Rep

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Due to their different specificity, the use of Vip3 proteins from Bacillus thuringiensis in combination with the conventionally used Cry proteins in crop protection is being essential to counteract the appearance of insect resistance. Therefore, understanding the mode of action of Vip3 proteins is crucial for their better application, with special interest on the binding to membrane receptors as the main step for specificity. Derived from in vitro heterologous competition binding assays using 125I-Vip3A and other Vip3 proteins as competitors, it has been shown that Vip3 proteins share receptors in Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). In this study, using 125I-Vip3Aa, we have first extended the in vitro competition binding site model of Vip3 proteins to Spodoptera littoralis. With the aim to understand the relevance (in terms of toxicity) of the binding to the midgut sites observed in vitro on the insecticidal activity of these proteins, we have performed in vivo competition assays with S. littoralis larvae, using disabled mutant (non-toxic) Vip3 proteins as competitors for blocking the toxicity of Vip3Aa and Vip3Af. The results of the in vivo competition assays confirm the occurrence of shared binding sites among Vip3 proteins and help understand the functional role of the shared binding sites as revealed in vitro.

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Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis

Cited by 7 publications

References 49 publications

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Domain III β4–β5 Loop and β14–β15 Loop of Bacillus thuringiensis Vip3Aa Are Involved in Receptor Binding and Toxicity

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

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