Essential role of amino acids in αD-β4 loop of a Bacillus thuringiensis Cyt2Aa2 toxin in binding and complex formation on lipid membrane

Bacillus thuringiensis subsp. darmstadiensis produces Cyt2Aa2 toxin that shows invivo specific toxicity against Dipteran insect larvae but exhibits invitro cytolytic activity to a broad-spectrum of cells including red blood cells. Two mutant toxins have been generated by introducing a small hydrophobic alanine into positions Thr-144 and Asn-145 in αD-β4 loop. Both mutants were highly expressed as crystalline inclusions that were solubilized in alkaline conditions and processed by chymotrypsin to yield activated products similar to that of the wild type protein. The T144A mutant shows lower hemolytic activity but exhibits larvicidal activity against Aedes aegypti and Culex quinquefasciatus larvae comparable to the wild type. In contrast, loss of mosquito-larvicidal and hemolytic activities was observed for the N145A mutant. Membrane interaction assays shows that the T144A mutant binds and forms complexes on liposomes, sheep red blood cells and brush border membrane fractions (BBMF) from A. aegypti larvae whereas the N145A mutant did not bind to these membranes. Our data suggested that amino acids in αD-β4 loop are important for specific binding and play a key role during toxin complex formation to lyse the targeted cell membranes.