RESUMO
Bacillus thuringiensis Cry4Ba mosquito-active toxin was previously shown to utilize two critical loop-residues, Tyr(332) and Phe(364) which are respectively located in ß2-ß3 and ß4-ß5 loops, for synergistic interactions with its alternative receptor-Cyt2Aa2. Here, structural analysis of the Cry4Ba-receptor-binding domain revealed that its N-terminal subdomain encompasses ß2-ß3 and ß4-ß5 hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr(328) in ß2 and Thr(369) in ß5. Functional importance of these two side-chains was demonstrated by single-Ala substitutions (T328A and T369A), adversely affecting toxin activity against Aedes aegypti larvae. Unlike toxicity restoration of the inactive E417A/Y455A toxin mutated within another receptor-binding subdomain, defective bioactivity of T328A and T369A mutants cannot be restored by Cyt2Aa2 as also observed for ß2-ß3 (Y332A) and ß4-ß5 (F364A) loop-mutants. ELISA-based analysis further verified a loss in binding of all four bio-inactive mutants (T328A, Y332A, T369A and F364A) to the immobilized Cyt2Aa2. Protein-protein docking suggested that the two critical loop-residues (Tyr(332) and Phe(364)) correspondingly located at ß2-ß3 and ß4-ß5 loops can clearly interact with four counterpart surface-exposed residues of Cyt2Aa2. Altogether, our present data demonstrate structural importance of Thr(328) and Thr(369) toward hydrogen-bonded stabilization of two receptor-binding hairpins (ß2-ß3 and ß4-ß5) for synergistic toxicity of Cry4Ba with Cyt2Aa2.