Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.
Crystal structures combined with biochemical data show that the delta-endotoxins from Bacillus thuringiensis are structurally poised towards large-scale, irreversible conformational changes that transform them from the soluble protein bound at the cell surface into a membrane-embedded form causing lysis of susceptible insect cells. Cry delta-endotoxins are made of a helix bundle, a beta-prism and a beta-sandwich. The conformational change involves an umbrella-like opening between the helix-4,5-hairpin and the remaining helices, and between the helical domain and the two sheet domains. Comparison of Cry1Ac structures with and without the bound receptor ligand GalNAc associates occupation of the high-affinity site on the beta-sandwich with an increase of temperature factors in the helical, pore-forming domain, which may indicate how receptor binding could trigger the required major conformational change. The structure of Cyt delta-endotoxins indicates that the surface helix hairpins must peel away to expose the beta-strands for membrane attack. Single amino acid substitutions in hinge residues or the core can restore activity following an inhibitory mutation.
To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.
The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml).
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