Autaipohn KaikaewChamras PromptmasChanan AngsuthanasombatMahidol UniversityBiophysics Institute for Research and Development (BIRD)2018-12-112019-03-142018-12-112019-03-142016-01-15Biochemical and Biophysical Research Communications. Vol.469, No.3 (2016), 698-703109021040006291X2-s2.0-84953635241https://repository.li.mahidol.ac.th/handle/123456789/43142© 2015 Elsevier Inc. All rights reserved. Bacillus thuringiensis Cry4Ba mosquito-active toxin was previously shown to utilize two critical loop-residues, Tyr332and Phe364which are respectively located in β2-β3and β4-β5loops, for synergistic interactions with its alternative receptor-Cyt2Aa2. Here, structural analysis of the Cry4Ba-receptor-binding domain revealed tha 2015 Elsevier Inc. All rights reserved.t its N-terminal subdomain encompasses β2-β3and β4-β5hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr328in β2and Thr369in β5. 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 β2-β3 (Y332A) and β4-β5 (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 (Tyr332and Phe364) correspondingly located at β2-β3and β4-β5loops can clearly interact with four counterpart surface-exposed residues of Cyt2Aa2. Altogether, our present data demonstrate structural importance of Thr328and Thr369toward hydrogen-bonded stabilization of two receptor-binding hairpins (β2-β3and β4-β5) for synergistic toxicity of Cry4Ba with Cyt2Aa2.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/j.bbrc.2015.11.115