Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Satō, Ryōichi

doi:10.1042/bsr20120113

Cited by 8 publications

(6 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This conclusion also agrees with the previously reported hypothesis that Cry1Aa binds to cadherin‐like receptors at multiple sites presented by several loops (Fujii et al. ). The current study demonstrates that loop 2 is one of the most effective regions for introducing Cry1Aa affinity maturation to cadherin‐like receptors for the purpose of directed evolution of the toxin.…”

Section: Discussionsupporting

confidence: 93%

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Endo

Kobayashi²,

Hoshino³

et al. 2014

MicrobiologyOpen

Self Cite

View full text Add to dashboard Cite

Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371WGLA374, 371WPHH374, 371WRPQ37425) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.

show abstract

Section: Discussionsupporting

confidence: 93%

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Endo

Kobayashi²,

Hoshino³

et al. 2014

MicrobiologyOpen

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mutant libraries have been constructed in specific areas of the 3D-Cry toxin using several molecular approaches such as degenerated primers [ 133 , 134 , 135 ], DNA shuffling [ 136 , 137 ], or using a previously constructed antibodies library [ 138 ]. All these libraries were screened for variants showing high binding affinity toward two of the most well-known receptors (cadherin like receptor and APN), and although an increase of binding affinity is not a guarantee for increased toxicity [ 134 , 135 ], some authors have managed to obtain enhanced 3D-Cry toxin variants compared to the parental toxin.…”

Section: “In Vitro Evolution” Of 3d-cry Toxins: An Historical Persmentioning

confidence: 99%

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

Vı́lchez

2020

Toxins

View full text Add to dashboard Cite

3D-Cry toxins, produced by the entomopathogenic bacterium Bacillus thuringiensis, have been extensively mutated in order to elucidate their elegant and complex mechanism of action necessary to kill susceptible insects. Together with the study of the resistant insects, 3D-Cry toxin mutants represent one of the pillars to understanding how these toxins exert their activity on their host. The principle is simple, if an amino acid is involved and essential in the mechanism of action, when substituted, the activity of the toxin will be diminished. However, some of the constructed 3D-Cry toxin mutants have shown an enhanced activity against their target insects compared to the parental toxins, suggesting that it is possible to produce novel versions of the natural toxins with an improved performance in the laboratory. In this report, all mutants with an enhanced activity obtained by accident in mutagenesis studies, together with all the variants obtained by rational design or by directed mutagenesis, were compiled. A description of the improved mutants was made considering their historical context and the parallel development of the protein engineering techniques that have been used to obtain them. This report demonstrates that artificial 3D-Cry toxins made in laboratories are a real alternative to natural toxins.

show abstract

“…These changes may suggest a variation in mutant toxicity, as in the study conducted by Fuji et al that highlights the importance of the α8 loop of the Cry1Ab toxin in the interactions of the toxin with the cadherin BT-R1 receptor. 30,31 This suggests that mutants may be potentiated concerning the binding to the receptor or, in another context, an improvement in additional interactions with target insects that have not been reported yet.…”

Section: Structural Analysis Of the Best Sequences Obtained Using Hiddenmentioning

confidence: 97%

“…Several studies state that loop 3 region of Cry toxins is directly related to the significant binding to cadherin BT-R1 receptors with Cry1Aa and Cry1Ab binding to the BtR receptors of Heliothis virescens. 30 Studies on mutagenesis in coding regions to loop 3 conducted by Pacheco et al 34 demonstrate the importance of this amino acid region, in which variations of toxicity could be correlated to changes made in Cry1Ab loop 3, ascribing that the lack of binding of toxins to the BBMV of M. sexta result in changes in oligomerization and affect the toxicity of the protein Almost all mutants show changes in domain III, except for S15pop1-1. Studies conducted by Burton et al reported reductions in the toxicity of Cry1Ac proteins when making changes in domain III through mutagenesis.…”

Section: Structural Analysis Of the Best Sequences Obtained Using Hiddenmentioning

confidence: 99%

Generation of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling

Pinzón-Reyes

Sierra-Bueno

Suárez-Barrera

et al. 2020

Evol Bioinform Online

View full text Add to dashboard Cite

Directed evolution methods mimic in vitro Darwinian evolution, inducing random mutations and selective pressure in genes to obtain proteins with enhanced characteristics. These techniques are developed using trial-and-error testing at an experimental level with a high degree of uncertainty. Therefore, in silico modeling of directed evolution is required to support experimental assays. Several in silico approaches have reproduced directed evolution, using statistical, thermodynamic, and kinetic models in an attempt to recreate experimental conditions. Likewise, optimization techniques using heuristic models have been used to understand and find the best scenarios of directed evolution. Our study uses an in silico model named HeurIstics DirecteD EvolutioN, which is based on a genetic algorithm designed to generate chimeric libraries from 2 parental genes, cry11Aa and cry11Ba, of Bacillus thuringiensis. These genes encode crystal-shaped δ-endotoxins with 3 conserved domains. Cry11 toxins are of biotechnological interest because they have shown to be effective as biopesticides for disease-spreading vectors. With our heuristic model, we considered experimental parameters such as DNA fragmentation length, number of generations or simulation cycles, and mutation rate, to get characteristics of Cry11 chimeric libraries such as percentage of population identity, truncation of variants obtained from the presence of internal stop codons, percentage of thermodynamic diversity, and stability of variants. Our study allowed us to focus on experimental conditions that may be useful for the design of in vitro and in silico experiments of directed evolution with Cry toxins of 3 conserved domains. Furthermore, we obtained in silico libraries of Cry11 variants, in which structural characteristics of wild Cry families were observed in a review of a sample of in silico sequences. We consider that future studies could use our in silico libraries and heuristic computational models, as the one suggested here, to support in vitro experiments of directed evolution.

show abstract

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Cited by 8 publications

References 42 publications

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

Generation of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling

Contact Info

Product

Resources

About