Structural requirements of the unique disulphide bond and the proline-rich motif within the α4–α5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa δ-endotoxin

Tapaneeyakorn, Satita; Pornwiroon, Walairat; Katzenmeier, Gerd; Angsuthanasombat, Chanan

doi:10.1016/j.bbrc.2005.03.006

Cited by 13 publications

(6 citation statements)

References 26 publications

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“…The loop connecting α 4 and α 5, which has been shown to be critically involved in forming pore [15], has low fluctuation, indicating that this loop is more rigid than other loops in Cry4Aa trimer. This rigidity has been shown to be due to a disulphide bond (Cys 192 -Cys 199 ) and a proline-rich motif (Pro 193 ProAsnPro 196 ) within the loop [42]. …”

Section: Resultsmentioning

confidence: 99%

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

2010

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Cry4Aa toxin is one of the highly specific mosquito-larvicidal proteins produced by the bacterium Bacillus thuringiensis subspecies israelensis. It is thought to form pores in the larval midgut membrane that cause membrane leakage and subsequent insect death. Therefore, Cry4Aa and other Cry toxins have been used as efficient and safe bacterial insecticides to control the disease-carrying mosquitoes such as Aedes, Anopheles, and Culex. However, we still do not clearly understand how Cry toxins kill mosquito-larvae at molecular details. Recent electron crystallographic images of Cry4Ba toxin, another toxin closely related to Cry4Aa toxin, have suggested that the protein forms trimer in aqueous solution and in lipid monolayer. Moreover, the unit cell of X-ray crystal structure of Cry4Ba toxin has been shown to be trimeric. In this study, we constructed the first full-atom structural model of Cry4Aa trimer using the trimeric unit cell structure of Cry4Ba toxin as a template and then used the methods of molecular dynamics (MD) and molecular mechanics combined with Poisson-Boltzmann and surface area (MM-PBSA) to show that the trimeric structure of Cry4Aa toxin is stable in 150 mM KCl solution on 10 ns timescale. The results reveal that Cry4Aa toxins use polar amino acid residues on α-helices 3, 4, and 6 to form trimer and suggest that the proteins form trimer to reduce their non-polar interactions with surrounding water. Based on the obtained trimeric structure of Cry4Aa toxins, we propose that pore formation of Cry toxins may involve a 90°-hairpin rotation during the insertion of their three α4-α5 hairpins into the membrane. This process may be mediated by water and ions.PACS Codes: 87.15.ap, 87.15.bk, 87.14.ep

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Section: Resultsmentioning

confidence: 99%

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

2010

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“…2) is an important determinant for toxicity, conceivably being involved in an interaction with lipid head groups for stabilising the oligomeric pore structure (Pornwiroon JBMB 2004) [ Fig. 2, left view) (Tapaneeyakorn BBRC 2005) [49]. Possibly, structural integrity of the 4-5 loop may indeed play an important role in the membrane insertion step.…”

Section: Insights Into the Mechanism Of Membrane Pore Formationmentioning

confidence: 99%

Mosquito Resistance to Bacterial Larvicidal Toxins

Wirth¹

2013

TOTNJ

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Abstract:The insecticidal character of the three-domain Cry -endotoxins produced by Bacillus thuringiensis during sporulation is believed to be caused by their capability to generate lytic pores in the target larval midgut cell membranes. This review describes toxic mechanisms with emphasis on the structural basis of pore formation by two closely related dipteran-specific toxins, Cry4Aa and Cry4Ba, which are highly toxic to mosquito larvae. One proposed toxic mechanism via an "umbrella-like" structure involves membrane penetration and pore formation by the 4-5 transmembrane hairpin. The lipid-induced -conformation of 7 could possibly serve as a lipid anchor required for an efficient insertion of the pore-forming hairpin into the bilayer membrane. Though current electron crystallographic data are still inadequate to provide such critical insights into the structural details of the Cry toxin-induced pore architecture, this pivotal evidence clearly reveals that the 65-kDa active toxin in association with the lipid membrane could exist in at least two different trimeric conformations, implying the closed and open states of a functional pore.

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“…To target this, the N546 was deleted (Δ) or displaced with the simplest amino acid, glycine (G), an acidic amino acid, aspartic acid (D) and a basic amino acid, lysine (K). SDS-PAGE analysis showed that mutants N546G, N546D and N546K well expressed 130 kD protoxin protein, but mutant N546Δ showed a clear reduction in protein amount corresponding to 130 kD band, accompanied by the presence of some bands of lower molecular weight, as can be interpreted as degradation products (Figure 2), which might A be structurally consistent to destruction of structural integrity of the loop containing N546, probably introducing structural disorder in the flanking β-sheets, thus leading to protein instability [33,36] . When tested in insect bioassays, mutant N546G had similar insecticidal activity to wild-type Cry1Ac but was less active than N546A, likely because this simplest amino acid at this position affected the main-chain conformation of this loop in contrast to alanine.…”

Section: Resultsmentioning

confidence: 94%

“…All residues in this loop were respectively substituted with alanine, and the resultant Cry1Ac mutant toxins were highly produced in acrystalliferous Cry − B. When equal amounts of cell lysates were analyzed using SDS-PAGE, except W544A and G545A that showed a reduced amount of 130-kD protein likely due to protein misfolding [33] , all mutants yielded a 130-kDa protoxin band at levels comparable to that of the wild-type toxin ( Figure 2). When each mutant toxin was tested for their relative toxicity against H. armigera larvae, mutants N543A, W544A, G545A, S547A, S548A and I549A exhibited varying degrees of reduced toxicity, but the mutant N546A showed almost 2 times increased activity than wild-type Cry1Ac toxin (Table 1).…”

Section: Resultsmentioning

confidence: 95%

The role of β18–β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

et al. 2008

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Structural requirements of the unique disulphide bond and the proline-rich motif within the α4–α5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa δ-endotoxin

Cited by 13 publications

References 26 publications

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

Mosquito Resistance to Bacterial Larvicidal Toxins

The role of β18–β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

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