Cys31, Cys47, and Cys195 in BinA are essential for toxicity of a binary toxin from Bacillus sphaericus.
The mosquito larvicidal binary toxin produced by Bacillus sphaericus is composed of 2 proteins called BinA and BinB. While BinB acts as specificity determinant, BinA is expected to bind to BinB, translocates into cytosol, and exerts its activity via an unknown mechanism. To study the role of cysteine in BinA, 3 cysteine residues were substituted by alanine and serine. Substitution at Cys195 significantly reduced the toxin activity, whereas substitution at Cys31 and Cys47 abolished its toxicity. Intrinsic fluorescent analysis suggested that all mutant proteins should have similar tertiary structure to that of the wild type. Analysis of the mutant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without a reducing agent indicated that all 3 cysteine residues were not involved in disulfide bond formation within the BinA molecule. This is the first report to demonstrate that cysteine residues at 3 positions in BinA are required for full toxicity of the binary toxin. They may play a critical role during oligomerization or interaction between BinA and BinB to form the active complex.
Promdonkoy
Boonhiang
B
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand. boonhiang@biotech.or.th
Promdonkoy
Patcharee
P
Wongtawan
Busabun
B
Boonserm
Panadda
P
Panyim
Sakol
S
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D003465:Culex / Q000187:drug effects; D003545:Cysteine / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007814:Larva / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016019:Survival Analysis
D001427:Bacterial Toxins; C046134:binB protein, Bacillus sphaericus; D003545:Cysteine