Bacillus thuringiensis Cry4A and Cry4B Mosquito-larvicidal Proteins: Homology-based 3D Model and Implications for Toxin Activity

Abstract

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B δ-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquitolarvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a threedomain organization: the N-terminal domain (I) is a sevenhelix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel β-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting α4 and α5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.

Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

“…The activated Cry4Aa and Cry4Ba are ~65 kDa toxins with three distinct-domains. The N-terminal domain I is a seven helix bundle responsible for pore formation, and the following two resemble carbohydrate binding proteins: a β-prism (domain II) and a plant lectin-like β-sandwich (domain III) [81,102,103,104,105]. Their three-dimensional structures are similar to the tertiary structures of other previously-solved Cry’s [106,107,108].…”

“…Consistent with the differential specificities, the identity between the amino acid sequences of their N-termini toxic portions is only about 30% (55% similarity) [80,81]. Cry11Aa is encoded by a sequence of 1929 bp (643 amino acids) and displays high larvicidities against the larvae of Aedes and Culex but lower against Anopheles [19,21,82].…”

Mosquito Resistance to Bacterial Larvicidal Toxins

“…Another trigger could be proteolytic cleavage in the solvent-exposed loops connecting helices in the bundle that could confer greater flexibility on the tertiary structure of the toxin molecule. Indeed, there is additional in vitro proteolysis occurring in the exposed loop linking 5 and 6 of both the 65-kDa activated Cry4Aa and Cry4Ba toxins, producing two non-covalently associated fragments of ~20 kDa and ~47 kDa which are mapped to the first five helices ( 1-5) and 6-7-linked domains II-III, respectively (Angsuthanasombat JBMB 2004) [33]. However, a discrepancy was observed between in vitro and in vivo toxicity results when a tryptic cleavage site in this loop of Cry4Ba was eliminated (Angsuthanasombat FML 1993) [34].…”

“…During the past several years, our research has focused on the molecular mechanism of toxicity of the two closely related mosquito-larvicidal proteins, Cry4Aa and Cry4Ba (Angsuthanasombat JBMB 2004) [33]. We now feel able to tackle some of the key steps viz the killing mechanism of these insecticidal proteins, particularly on the events following insertion of the 4-5 hairpin of the three-domain activated toxin into the lipid membrane, resulting in the formation of ion-leakage pores.…”

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

“…In spite of this variability, some similarity has been found in the three-domain Cry proteins, evidenced by the presence of three conserved domains in the tertiary structure of the active toxin (delta-endotoxin), even from Cry toxins showing low levels of identity (i.e., the Cry1-type, Cry3-type, and Cry4-type toxins): one domain with a bundle of ␣ helices and two domains of ␤ sheets (3). This uniformity is, at least in part, a reflection of the five conserved blocks in the gene structure, which are present in almost all the cry genes (10).…”