Isoleucine at position 150 of Cyt2Aa toxin from Bacillus thuringiensis plays an important role during membrane binding and oligomerization

Abstract

Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin producedby Bacillus thuringiensis subsp. darmstadiensis. The toxin becomesinactive when isoleucine at position 150 was replacedby alanine. To investigate the functional role of this position,Ile150 was substituted with Leu, Phe, Glu and Lys. All mutantproteins were produced at high level, solubilized in carbonatebuffer and yielded protease activated product similar to thoseof the wild type. Intrinsic fluorescence spectra analysis suggestedthat these mutants retain similar folding to the wildtype. However, mosquito larvicidal and hemolytic activitiesdramatically decreased for the I150K and were completelyabolished for I150A and I150F mutants. Membrane bindingand oligomerization assays demonstrated that only I150E andI150L could bind and form oligomers on lipid membrane similarto that of the wild type. Our results suggest that amino acidat position 150 plays an important role during membranebinding and oligomerization of Cyt2Aa2 toxin. [BMB Reports2013; 46(3): 175-180]

Keywords

• Bacillus thuringiensis
• Cyt toxin
• Membrane binding
• Mutagenesis
• Oligomerization