Role of cysteine at positions 67, 161 and 241 of a Bacillus sphaericus binary toxin BinB

Abstract

Binary toxin consisting of BinA and BinB from Bacillus sphaericus is toxic to mosquito larvae. BinB is responsible for specific binding to the larval gut cell membrane while BinA is crucial for toxicity. To investigate functional role of cysteine in BinB, three cysteine residues at positions 67, 161, and 241 were replaced by alanine or serine. Mutations at these positions did not affect protein production and overall structure of BinB. These cysteine residues are not involved in disulfide bond formation between BinB molecules. Mosquito-larvicidal assays revealed that C67 and C161 are essential for toxicity, whereas C241 is not. Mutations at C67 and C161 resulted in weaker BinA-BinB interaction. The loss of toxicity may be due to the reduction of interactions between BinA and BinB or BinB and its receptor. C67 and C161 could also play a part during conformational changes or internalization of the binary toxin into the target cell. [BMB reports 2010; 43(1): 23-28]

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

“…Since the interaction of the binary toxin with target lipid membrane is one of the key steps in eliciting cytopathological effects on mosquito larvae, molecular insights into the interaction of the binary toxin with lipid membranes is required to gain a better understanding of the mechanism of toxicity. Due to the lack of three dimensional structure of the binary toxin, functional characterization has been based mainly on its amino acid sequence analysis and secondary structure prediction (10)(11)(12)(13)(14)(15). Aromatic residues, especially tyrosine and tryptophan, conceivably play a key role in membrane anchoring of many membrane proteins and are mainly found at the membrane-water interface (16).…”

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

“…The recombinant plasmid pET-tBinB (Boonyos et al, 2010) encoding the 43 kDa active fragment of BinB wild-type toxin amino 0022-2011/$ -see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.10.004 acid positions Asn33-Lys408, which was cloned from B. sphaericus 2297 into NheI and XhoI sites of pET-17b was used as a template for PCR with appropriate primers shown in Table 1.…”

“…Whereas all larvae were killed (100% mortality) when fed with mixture of wild-type BinA and BinB, toxicity was significantly reduced when BinB was replaced by individual BinB constructs (Table 2). LC 50 (concentration of toxin that cause 50% mortality) of the mixture of BinA and BinB inclusions is normally in the range of 10-100 ng/ml when using the full-length or active truncated toxin (Boonyos et al, 2010;Singkhamanan et al, 2010).…”

“…doi:10.1016/j.jip.2010.10.004 acid positions Asn33-Lys408, which was cloned from B. sphaericus 2297 into NheI and XhoI sites of pET-17b was used as a template for PCR with appropriate primers shown in Table 1. This truncated BinB still retained full activity similar to that of the full-length BinB (Boonyos et al, 2010). Plasmids encoding BinB N-terminal constructs, N-11K (N33-F132), N-25K (N33-K254) and N-32K (N33-R318), were generated based on Stratagene QuikChange™ Site-directed mutagenesis by substitutions of codons encoding E133, M255 and S319 with stop codons, respectively.…”

“…Plasmids encoding the respective C-terminal fragments were constructed by PCR. The plasmid pET-tBinB (Boonyos et al, 2010) was used as a template together with T7 terminator primer and appropriate forward primers (Table 1) designed for encoding different C-terminal BinB constructs, C-32K (E133 to K408), C-18K (M255 to K408) and C-11K (S319 to K408). PCR products were digested with NdeI and XhoI and cloned into pET-17b that was digested with the same enzymes.…”