Bacillus thuringiensis Cry4Ba toxin employs two receptor-binding loops for synergistic interactions with Cyt2Aa2

• Bti-Cry4Ba employs two receptor-binding loops for synergism with Btd-Cyt2Aa2.
• Cry4Ba–Cyt2Aa2 synergistic interactions were revealed through toxicity restoration.
• Aromaticity of β2–β3 loop-Y322 and β4–β5 loop-F364 is implicated in receptor-binding.
• Cry4Ba utilizes Tyr332 and Phe364 for synergistic interactions with Cyt2Aa2.

We previously demonstrated that co-expression in Escherichia coli of Bacillus thuringiensis (Bt) subsp. israelensis Cry4Ba and Bt subsp. darmstadiensis Cyt2Aa2 shows high synergistic toxicity against target mosquito larvae. Here, further insights into synergistic interactions between these two toxins were revealed through bioactivity restoration of particular inactive Cry4Ba-mutant toxins altered within the receptor-binding domain. Specific mutations at β2–β3 (Y332A) or β4–β5 (F364A) loops, but neither at three other β-hairpin loops (β6–β7, β8–β9 and β10–β11) of Cry4Ba, adversely affect toxicity restoration by Cyt2Aa2. Binding analysis using quartz crystal microbalance verified a decrease in binding of these two bioinactive-mutant toxins (Y332A and F364A) to the immobilized Cyt2Aa2. This suggests that Cry4Ba utilizes these two critical aromatic loop-residues, Tyr332 and Phe364, for synergistic toxicity with its alternative receptor-Cyt2Aa2.