Structure of the insecticidal bacterial δ-endotoxin Cry3Bb1 ofBacillus thuringiensis

Abstract

The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two delta-endotoxins.

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

“…The crystal structures of several trypsin-activated Cry proteins (Cry1Aa, Cry3Aa, Cry3Bb and Cry2Aa) have been elucidated [6][7][8][9], revealing a similar three-domain structure. The N-terminal domain I consists of seven α-helices, and is thought to be responsible for toxin insertion into the cell membrane, leading to pore formation.…”

Mosquito Resistance to Bacterial Larvicidal Toxins

“…Thus far, the three-dimensional structures of Bt Cry toxins have been determined by X-ray crystallography in almost all the major specificity classes, including the lepidoteran-specific Cry1Aa (PDB code: 1CIY) (Grochulski JMB 1995) [15], the lepidopteran/dipteran-dual specific Cry2Aa (PDB code: 1I5P) (Morse Structure 2001) [16], the coleopteran-specific Cry3Aa (PDB code: 1DLC) (Li Nature 1991) [17] and Cry3Bb (PDB code: 1JI6) (Galitsky ACD 2001) [18], the dipteran-specific Cry4Aa (PDB code: 2C9K) (Boonserm J Bacteriol 2006) [19] and Cry4Ba (PDB code: 1W99) (Boonserm JMB 2005) [20], and more recently another coleopteran-specific Cry8Ea (PDB code: 3EB7) (Guo JSB 2009) [21]. Undoubtedly, all these known structures have been a valuable contribution to the Bt research area since they have been providing a greater understanding for the structural basis of their insect specificity and gut epithelial cell lysis.…”

Susceptibilidade de larvas de Cerotoma arcuata Olivier (Coleoptera: Chrysomelidae) a Beauveria bassiana (Bals.) Vuillemin, Metarhizium anisopliae (Metsch.) Sorokin e Bacillus thuringiensis Berliner

“…às toxinas de B. thuringiensis é amplamente estudada. Experimentos avaliam a expressão de novas delta-endotoxinas em milho transgênico (MOELLENBECK et al, 2001;AL-DEEB & WILDE, 2005), variações mais letais das toxinas (VAUGHN et al, 2005), mecanismos responsáveis pela seletividade da toxina para D. undecimpunctata (GALITSKY et al, 2001), assim como os efeitos em insetos não-alvos (AL-DEEB & WILDE, 2003). Esses exemplos indicam que a faixa neutra de pH do intestino das larvas de C. arcuata não impede que o inseto seja controlado com o patógeno B. thuringiensis, como verificado neste experimento pela mortalidade causada pela estirpe CG 940.…”