De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Tetreau, Guillaume; Sawaya, M.R.; Zitter, E. De; Андреева, Е.Р.; Banneville, Anne‐Sophie; Schibrowsky, Natalie A.; Coquelle, Nicolas; Brewster, Aaron S.; Grünbein, Marie Luise; Kovács, Gabriela Nass; Hunter, Mark S.; Kloos, Marco; Sierra, Raymond G.; Schirò, Giorgio; Qiao, Pei; Stricker, Myriam; Bideshi, Dennis K.; Young, I.D.; Zala, Ninon; Engilberge, Sylvain; Gorel, A.; Signor, Luca; Teulon, Jean‐Marie; Hilpert, M.; Foucar, L.; Bielecki, Johan; Bean, Richard; Wijn, Raphaël de; Sato, Tokushi; Kirkwood, Henry; Letrun, Romain; Batyuk, A.; Fenel, Daphna; Schubert, Robin; Canfield, Ethan; Alba, Mario M.; Laporte, Fréderic; Després, Laurence; Bacia, Maria; Roux, Amandine Le; Chapelle, Christian; Riobé, François; Maury, Olivier; Ling, Wai Li; Boutet, Sébastien; Mancuso, Adrian P.; Gutsche, Irina; Girard, Éric; Barends, Thomas R. M.; Pellequer, Jean‐Luc; Park, Hyun-Woo; Laganowsky, Arthur; Rodríguez, José A.; Burghammer, Manfred; Shoeman, Robert L.; Doak, R. Bruce; Weik, Martin H.; Sauter, Nicholas K.; Federici, Brian A.; Cascio, Duilio; Schlichting, Ilme; Colletier, J.P.

doi:10.1038/s41467-022-31746-x

Cited by 16 publications

(14 citation statements)

References 104 publications

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“…Values obtained from all these profiles are assembled in distribution plots (Figure 1G). Taken together, the size of Cry11Aa monomers was estimated as (15.5 ± 1.1) × (15.5 ± 4.1) nm 2 , which is very close to that obtained from the 3D crystal structure, 6 × 16 × 17 nm 3 , by the x‐ray crystallography 26 …”

Section: Resultssupporting

confidence: 82%

“…[23][24][25][26][27][28] As described above, the two proteolytically cleaved Cry11Aa fragments remain associated, 7 and thus, the 3D structure of active Cry11Aa must resemble the crystal structure of the protoxin. 26 Since multimerization of Cry11Aa molecules is a key process for its toxic activity, 22 the native association of Cry11Aa monomers in crystal is likely most representable for the nature of the protein assembled in the process of toxic actions. Despite the 3D crystal structure of Cry11Aa (or Cyt1Aa) protoxin has been determined, 26,28 the molecular arrangement of the protein crystal subjective to the native growth is difficult for visual perception of the lattice pattern of crystal cell units.…”

mentioning

confidence: 94%

mentioning

confidence: 99%

“…Atomic force microscopy (AFM) has been widely applied to visualizations of the distribution and multimerization of Cry and Cyt toxins from various Bt subspecies interacting with different membrane systems 23–28 . As described above, the two proteolytically cleaved Cry11Aa fragments remain associated, 7 and thus, the 3D structure of active Cry11Aa must resemble the crystal structure of the protoxin 26 . Since multimerization of Cry11Aa molecules is a key process for its toxic activity, 22 the native association of Cry11Aa monomers in crystal is likely most representable for the nature of the protein assembled in the process of toxic actions.…”

mentioning

confidence: 99%

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Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography

Chen

Teulon

Pellequer

2023

J of Molecular Recognition

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Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L‐weight filter to enhance the structural features. By L‐weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three‐dimensional structures determined by x‐ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions.

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Section: Resultssupporting

confidence: 82%

mentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography

Chen

Teulon

Pellequer

2023

J of Molecular Recognition

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“…A previous study revealed the 3D structures of a long Cry1Ac (130 kDa) and short Cry2Aa protoxin (70 kDa), showing that the Cry1Ac protoxin consists of seven domains (21) (Figure 1), whereas the Cry2Aa protoxin has a three-domain organization (22). The 3D structural data of several activated Cry toxins showed a similar structure to the short protoxin, consisting of three domains, which also form the first three domains of the long protoxin (23)(24)(25)(26)(27)(28)(29)(30)(31)(32) (Figure 1). Domain I contains seven a-helices and it is involved in oligomerization and membrane insertion, whereas domains II and III both consist mainly of b-sheets structures and are involved in receptor binding and specificity (6,10).…”

Section: Cry Pfts Produced By Btmentioning

confidence: 94%

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis

et al. 2023

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Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

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Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Takahashi,

Asakura,

Ide

et al. 2024

Curr Microbiol

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Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

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De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cited by 16 publications

References 104 publications

Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography

Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

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