In Silico Structure-Based Identification and Validation of Key Residues of Vip3Aa Involving in Lepidopteran Brush Border Receptor Binding

Chi, Baoyan; Li, Haitao; Zhang, Jinbo; Wei, Panpan; Gao, Jiguo; Liu, Rongmei

doi:10.1007/s12010-018-2880-6

Cited by 8 publications

(11 citation statements)

References 22 publications

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“…Domain 3 of 3D-Cry proteins has been implicated in target specificity and glycan binding in previous studies 24 . The role of Vip3 domains 4 and 5 in target insect specificity may be supported by other studies that showed mutation of residues in domain 4 of Vip3Aa11 altered toxicity levels against Spodoptera exigua and Helicoverpa armigera 25 and alanine scanning mutagenesis of the Vip3Af1 protein identified a number of mutants in this region that caused significant loss of activity against Spodoptera frugiperda and Agrotis segetum 26 . Furthermore, DALI analysis of domains 4 and 5 of Vip3Bc1 identified a number of structural homologues of these domains with putative and demonstrated glycan binding capabilities 27 .…”

Section: Discussionmentioning

confidence: 71%

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane

et al. 2021

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Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin–membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.

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Section: Discussionmentioning

confidence: 71%

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane

et al. 2021

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“…Alan (26) nded that after a series of calculation rules, it predicts the amino acid sites that may form disul de bonds in protein molecules, and the prediction accuracy is 99.4%. Therefore, we combined the conserved structure region of Sip1Aa, Swiss pdbviewer (22) , and Laplace conformation to design a disul de bond mutation to study the effect on Sip1Aa-related functions. Finally, we selected four pairs of sites for mutations, introduced new disul de bonds, successfully express 37.6 kDa soluble protein, indicating that these amino acid residues were replaced by alanine without destroying the advanced structure of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…Using an online analysis server (http://swissmodel.expasy.org/), twenty pairs of disul de bond-forming sites were predicted. Seven pairs of sites were screened out based on the Sip1Aa conserved region, Swiss pdbviewer (22) , and Laplace conformation map. A pair of amino acid sites with a spacing of less than 50 bp was excluded, and eight cysteine mutation sites were determined, The pET-Sip1Aa plasmid was used as the template for site-directed mutagenesis, and eight pairs of mutant primers T149C-F/R251C-R, T149C-R/R251C-F, G153C-F/H248C-R, G153C-R/H248C-F, T158C-F/K243C-R, T158C-R/K243C-F, K178C-F/G314C-R, and K178C-R/G314C-F were used, respectively (the sequences were shown in Primersandaminoacidsequences in the additional materials) for PCR ampli cation according to the instructions in the site-directed mutagenesis kit, and 5 µL of the PCR products were used for agarose gel electrophoresis detection, and the electrophoresis results were shown in Fig.…”

Section: Prediction Of Disul De Key Pointmentioning

confidence: 99%

Improved structure and enhanced the insecticidal activity of Sip1Aa protein by adding disulfide bonds

Lin¹,

Wang²,

Ding³

et al. 2021

Preprint

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Sip1Aa is an insecticidal protein of Bacillus thuringiensis at the secretory stage. It has a strong toxic effect on the members of order Coleoptera. To date, there are few available studies on Sip1Aa protein and the inclusion body problem is serious, and this raises the importance to conduct further studies on Sip1Aa protein. Disulfide bonds, as the only covalent bond on protein side chains, play an important role in the stability and function of the proteins. The tertiary structure of Sip1Aa protein was analyzed by homologous modeling and other bioinformatics methods to predict the conserved domain of Sip1Aa protein. Cysteine used to replace these amino acids by site-directed mutation. Consequently, we were able to successfully construct Sip149-251, Sip153-248, Sip158-243, and Sip178-314. These were exposed to ultraviolet radiation, and we found that Sip153-248 and Sip158-243 were the most stable, followed by Sip149-251 and Sip178-314 when compared with Sip1Aa. After the mutant strain was transferred into Escherichia coli BL21, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect the inducible expression products. Approximately 37.6 kDa of proteins that were highly expressed in E. coli. We found no significant change in the insecticidal activity.

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“…The Sip1Aa protein (undisclosed) was discovered in our laboratory. According to Chi et al (2018), the amino acid sequence of Sip1Aa was submitted to the automated protein structure homology-modeling server (Phyre 2 ) 1 and run in intensive mode to generate the multi-template modeling (Kelley et al, 2015). The resulting 3D model was submitted to the ModRefiner server 2 for optimization, and then the model was evaluated by RAMPAGE 3 .…”

Section: Computer-aided Modeling Of the Three-dimensional Of Sip1aa Pmentioning

confidence: 99%

“…Ward et al (1988) mutated acidic and basic amino acids into alanine to explore its role in polar phospholipid group interaction. Chi et al (2018) and Luo et al (2018) applied site-directed mutagenesis in the study of key sites of the insecticidal activity of Vip3Aa11 protein. Yang et al (2014) applied site-directed mutagenesis in the study of improving the activity and stability of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

In silico Structure–Based Investigation of Key Residues of Insecticidal Activity of Sip1Aa Protein

et al. 2020

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Colaphellus bowringi Baly mainly damages cruciferous vegetables, leading to huge economic losses. The secretory insecticidal protein (Sip) of Bacillus thuringiensis (Bt) has high insecticidal activity against C. bowringi Baly. The tertiary structure of Sip1Aa protein was analyzed by homologous modeling and other bioinformatics methods to predict the conserved domain of Sip1Aa protein. Acidic and basic amino acids in the conserved domain were selected, and alanine was used to replace these amino acids by site-directed mutation. The difference between the insecticidal activities of mutant protein and Sip1Aa protein was analyzed. The insecticidal activities of H99A, K109A, K128A, and E130A against C. bowringi Baly were significantly increased, among which that of K128A was the most obviously changed, and the LC 50 value was decreased by about 10 times compared with that of Sip1Aa protein. The LC 50 value of mutant E130A was 0.286 µg/mL, which was about six times less than that of Sip1Aa. K128 and E130 were both in the β9-β10 loop. The toxicity of D290A, H242A, and H303A to C. bowringi Baly was significantly reduced, and their LC 50 value increased by about six, eight, and three times compared with that of Sip1Aa protein, respectively. This study showed that acidic and basic amino acid residues played a certain role in the toxicity of Sip1Aa protein, and the loss of side chains in key residues had a significant impact on the insecticidal activity of the protein. This study provides the theoretical basis for revealing the relationship between the structure and function of Sip1Aa protein and also provides a new method for the subsequent study of sip gene.

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In Silico Structure-Based Identification and Validation of Key Residues of Vip3Aa Involving in Lepidopteran Brush Border Receptor Binding

Cited by 8 publications

References 22 publications

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane

Improved structure and enhanced the insecticidal activity of Sip1Aa protein by adding disulfide bonds

In silico Structure–Based Investigation of Key Residues of Insecticidal Activity of Sip1Aa Protein

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