Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture

Thamwiriyasati, Niramon; Kanchanawarin, Chalermpol; Imtong, Chompounoot; Chen, Chun‐Jung; Li, Hui-Chun; Angsuthanasombat, Chanan

doi:10.1016/j.bbrc.2022.06.065

Cited by 5 publications

(7 citation statements)

References 39 publications

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“…Other studies indicated that the helix α-4 participates in oligomerization, as mutation Cry1AcN135Q affected oligomerization and toxicity against M. sexta ( 44 ). In addition, based on the Cry4Ba three-dimensional structure, a model simulating the insertion of the α-4–α-5 hairpin was constructed with the aim of increasing understanding of the pore’s possible structure ( 45 ).…”

Section: Cry Pfts Produced By Btmentioning

confidence: 99%

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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Section: Cry Pfts Produced By Btmentioning

confidence: 99%

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

et al. 2023

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“…The high-resolution structure of several Cry toxins has been elucidated in their activated, three-domain form, e.g., Cry3A [13], Cry4Ba [14,15], Cry4Aa [16], Cry1Aa [17], Cry2Aa [18], Cry3Bb1 [19], Cry8Ea1 [20], Cry5B [21,22], Cr7Ca1 [23], Cry1Da [24] and Cry11 (Aa and Ba) [25]. In all cases, the N-terminal domain I is formed by seven α-helices, α1 to α7 [13], and is involved in oligomerization and pore formation [14,16], whereas domains II and III contain mostly β-strands and are important for binding to receptors and in structural integrity [26].…”

Section: Structure Of Cry Toxinsmentioning

confidence: 99%

Channel Formation in Cry Toxins: An Alphafold-2 Perspective

Torres,

Surya,

Boonserm

2023

IJMS

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Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an ‘extended model’ where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the ‘membrane-inserted’ structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

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“… Combined surface-ribbon representations of the 65-kDa crystal structures of two closely related Cry4 mosquito-active toxins, illustrating their three-domain organization (DI-DIII). ( A ) Cry4Aa-R235Q (PDB: 2C9K [ 13 ]) and ( B ) Cry4Ba-R203Q (PDB: 4MOA [ 14 ]), Insets, zoomed in views of potential receptor-binding residues, which are situated in each different domain (Cry4Aa-DII and Cry4Ba-DIII) are represented as ball-and-stick models. …”

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Currently, the X-ray crystal structures of numerous Cry toxins [ 9 , 10 , 11 , 12 , 13 , 14 ], including the two mosquito-active toxins—Cry4Aa and Cry4Ba [ 13 , 14 ] have been solved. All these 65-kDa activated toxins display a typical wedge-shaped arrangement of three structurally different domains: an N-terminal domain of anti-parallel α-helical bundle(DI), a middle domain of three-β-sheet prism (DII) and a C-terminal domain of β-sheet sandwich (DIII) [ 13 , 14 ] (see Figure 1 ). Despite the fact that both Cry4Aa and Cry4Ba structures bear a resemblance to the other known Crystructures, their finer features are rather different since there is additional in vitro proteolysis by trypsin occurring at Cry4Aa-Arg 235 as well as Cry4Ba-Arg 203 located in the α5-α6 loop within DI [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

Dechkla

Charoenjotivadhanakul

Imtong³

et al. 2022

Toxins

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The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culexmosquito larvae than its closely related toxin—Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed inSpodoptera frugiperda(Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that bothCq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP—a Cry4Ba toxin receptor.In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALPexpressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain—DII, including Thr512, Tyr513 and Lys514 in the b10-b11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain—DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected b10-b11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatuslarvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position.Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform—Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

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Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture

Cited by 5 publications

References 39 publications

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

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

Channel Formation in Cry Toxins: An Alphafold-2 Perspective

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

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