The Role of β20–β21 Loop Structure in Insecticidal Activity of Cry1Ac Toxin from Bacillus thuringiensis

Liu, Yuan; Tang, Ying; Zhang, Yunlei; Xia, Liqiu; Fa-Xiang, Wang; Ding, Xuezhi; Yi, Siming; Yin, Jia

doi:10.1007/s00284-010-9760-9

Cited by 9 publications

(8 citation statements)

References 33 publications

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“…In the Cry1Ac toxin, the region corresponding to the Cry7Ca1 β15–β16 loop (Residues 542–549) on one the side of the β‐sheets was shown to participate in the sugar‐mediated receptor binding, while the region corresponding to the β21–β22 loop (Residues 608–616) on the opposite side of the β‐sheets was shown to affect toxicity against Helicoverpa armigenra larvae (Fig. ) …”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis

Jing

Yuan

et al. 2018

Protein Science

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Insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are widely used as environmentally friendly insecticides. As the only known Cry protein with insecticidal activity against Locusta migratoria manilensis, a locust subspecies that causes extensive destruction of crops, the Cry7Ca1 protein from Bt strain BTH‐13 identified in our previous study is of particular interest to locust prevention and control. However, the three‐dimensional structure of Cry7Ca1 toxin (the active form of the Cry7Ca1 protein) and the mechanisms of the Cry7Ca1 insecticidal specificity remain largely elusive. Here, we report a 2.3 Å crystal structure of the Cry7Ca1 toxin and carry out a systematic comparison of all available Cry toxins structures. A cluster of six loops in Cry toxin domain II, named Apex here, are the most variable structural elements and were documented to contribute in insecticidal specificity. The Cry7Ca1 toxin Apex loops are different from those of other Cry toxins in length, conformation, and sequence. Electrostatic potential analysis further revealed that Cry7Ca1 is the only structure‐available Cry toxin that does not have a high contrast of surface electrostatic potentials in the Apex. We further suggest that the L1/L2 loops in the center of the Cry7Ca1 Apex may be worthy of attention in future efforts to unravel the Cry7Ca1 insecticidal specificity as they exhibit unique features not found in the corresponding regions of other Cry toxins. Our work highlights the uniqueness of the Apex in the Cry7Ca1 toxin and may assist exploration of the insecticidal mechanism of the Cry7Ca1 against Locusta migratoria manilensis.

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Section: Resultsmentioning

confidence: 99%

“…2). 29 The Cry7Ca1 apex and its L1/L2 loop pair are very unique and deserve attention in studying Cry7Ca1 insecticidal specificity…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis

Jing

Yuan

et al. 2018

Protein Science

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“…The R508G mutation is located in a polymorphic region near β-sheet 18, which is the fourth conserved region among Cry toxins [89]. This region is rich in arginine and tyrosine residues and has been reported to interact with GPI-APN and GPI-ALP in the presence (as in Cry1Ac) or absence of Gal-NAc (as in the Cry toxins studied in this report) [34,99]. Therefore, mutations in this loop may likely affect interactions with receptors and result in an increase in flexibility due to the presence of a glycine residue flanking β-sheet 18 at position 508.…”

Section: In Silico Analyses Of Cry and Mutant Toxins For The Contrmentioning

confidence: 99%

“…However, in Cry8Ka5, the negative charge of glutamate (E538) in this region disrupts the interaction with D520 (Figure 7). In the loops of the three Domains, some residues have been characterized as important for Cry protein toxicity due to their involvement in the interaction with insect receptors [72,87,99,100]. Domain III is the smallest of the three Domains and has been reported to function as a receptor binding site for GPI-ALP, GPI-APN [41,65] and Gal-NAc [31].…”

Section: In Silico Analyses Of Cry and Mutant Toxins For The Contrmentioning

confidence: 99%

Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

Lucena

Pelegrini

Martins-de-Sa

et al. 2014

Toxins

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Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

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“…As for Cry proteins from B. thuringiensis, specific substitutions of Asn166 within the ␣4-␣5 loop in domain I of Cry4B almost completely abolished the toxicity against mosquito larvae, suggesting that the polarity at Asn166 plays a crucial role in the larvicidal activity (23). The residues Asn576 and Asn584 in ␤20-␤21 loop of the domain III of Cry1Ac were demonstrated to be on the inside of the molecular and considered critical to the stability of the protein structure (29).…”

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confidence: 99%

Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

Wang

Liu

Zhang

et al. 2012

Appl Environ Microbiol

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The crystal proteins from Bacillus thuringiensis are widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferous B. thuringiensis strain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host's intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.

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The Role of β20–β21 Loop Structure in Insecticidal Activity of Cry1Ac Toxin from Bacillus thuringiensis

Cited by 9 publications

References 33 publications

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis

Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

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