Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution

Abstract

The Cry4Aa ␦-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 Å. Like other Cry ␦-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-␣-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a ␤-prism (domain II) and a plant lectin-like ␤-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.

Mosquito Resistance to Bacterial Larvicidal Toxins

“…The N-terminal domain is a group of eight helices 1, 2a, 2b, 3, 4, 5, 6 and 7, initially assigned by Ellar group (Li Nature 1991) [17] in which the most hydrophobic helix ( 5) is surrounded by seven outer helices. The central helix is in fact not entirely hydrophobic, but rather exhibits an amphipathic character, as all of its polar or charged side-chains in the interhelical space are engaged in hydrogen bonds or salt bridges (Grochulski JMB 1995) [15] (Li Nature 1991) [17] (Boonserm J Bacteriol 2006) [19] (Boonserm JMB 2005) [20]. This is also the case for all the outer helices which are oriented with their polar or charged residues forming the outer surface of the helical bundle (Grochulski JMB 1995) [15] (Li Nature 1991) [ [27] have also been shown to be involved in receptor binding.…”

“…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.…”

“…3, 4, 5, 6 and 7, are long enough (>30Å) to span the lipid membrane. However, the hydrophobic faces of the outer-amphipathic helices of this pore-forming domain face inwards (Grochulski JMB 1995) [15] (Li Nature 1991) [17] (Boonserm J Bacteriol 2006) [19]. Therefore, these three-domain Cry toxins must undergo a major conformational change to convert the pore-forming apparatus into an aqueous transmembrane pore in which the hydrophobic surfaces would be in intimate contact with the membrane lipids.…”

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

“…The three dimensional structures of six Cry toxins with different insect specificities have been solved [2,3,9,15,22,26]. These toxins are composed of three domains -domain I, a seven α-helix bundle involved in membrane insertion, oligomer formation and pore formation [4]; domain II, a three anti-parallel β-sheets packed around a hydrophobic core in a "beta-prism" involved in receptor interaction [4]; and domain III, a β-sandwich of two antiparallel β-sheets also involved in receptor interaction [4].…”

Modelos caracterizando a interação entre as toxinas da família Cry1A de Bacillus thuringiensis e o receptor BT-R1 de Manduca sexta

“…As formas ativas de proteína são: Cry1Aa (Grochulski, P. et al, 1995), Cry1Ac (Derbyshire, D.J. et al, 2013), Cry3Bb (Galitsky, N. et al, 2001), Cry4Ba (Boonserm, P. et al, 2005), Cry4Aa (Boonserm, P. et al, 2006), Cry8Ea (Guo, S. et al, 2009), Cry5B (Hui, F. et al, 2012) Portanto, próCry1Ac trata-se de uma protoxina extensa e Cry2Aa de uma protoxina curta, muito similar às toxinas ativas (Tabela 4). Apesar da diferença na identidade de resíduos de aminoácidos (<45%) quando comparamos famílias 3D-Cry de primeira categoria diferentes, i.e.…”