Receptor binding is a prerequisite process to exert the mosquitocidal activity of the Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis. The beta-sheet prism (domain II) and beta-sheet sandwich (domain III) of the Cry4Ba toxin have been implicated in receptor binding, albeit the precise binding mechanisms of these remain unclear. In this work, alanine scanning was used to determine the contribution to receptor binding of some aromatic and hydrophobic residues on the surface of domains II and III that are predicted to be responsible for binding to the Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP) receptor. Larvicidal activity assays against A. aegypti larvae revealed that aromatic residues (Trp327 on the β2 strand, Tyr347 on the β3-β4 loop, and Tyr359 on the β4 strand) of domain II were important to the toxicity of the Cry4Ba toxin. Quantitative binding assays using enzyme-linked immunosorbent assay (ELISA) showed similar decreasing trends in binding to the Aa-mALP receptor and in toxicity of the Cry4Ba mutants Trp327Ala, Tyr347Ala, and Tyr359Ala, suggesting that a possible function of these surface-exposed aromatic residues is receptor binding. In addition, binding assays of the Cry4Ba toxin to the mutants of the binding residues Gly513, Ser490, and Phe497 of the Aa-mALP receptor supported the binding function of Trp327, Tyr347, and Tyr359 of the Cry4Ba toxin, respectively. Altogether, our results showed for the first time that aromatic residues on a side surface of the Cry4Ba domain II function in receptor binding. This finding provides greater insight into the possible molecular mechanisms of the Cry4Ba toxin.
Keywords: Aedes aegypti; Bacillus thuringiensis; Cry4Ba toxin; dengue fever; receptor binding.