title: Importance of Thr(328) and Thr(369) for functional maintenance of two receptor-binding β-hairpins of the Bacillus thuringiensis Cry4Ba toxin: Implications for synergistic interactions with Cyt2Aa2.
abstract: 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 β2-β3 and β4-β5 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 β2-β3 and β4-β5 hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr(328) in β2 and Thr(369) in 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 (Tyr(332) and Phe(364)) correspondingly located at β2-β3 and β4-β5 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 (β2-β3 and β4-β5) for synergistic toxicity of Cry4Ba with Cyt2Aa2.
authors.affiliation: Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand.
authors.forename: Autaipohn
authors.initials: A
authors.lastname: Kaikaew
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand.
authors.forename: Chamras
authors.initials: C
authors.lastname: Promptmas
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok, 10160, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000187:drug effects* / Q000502:physiology; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000008:administration & dosage* / Q000737:chemistry* / Q000648:ultrastructure; D001665:Binding Sites; D004357:Drug Synergism; D004731:Endotoxins / Q000008:administration & dosage* / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000008:administration & dosage* / Q000737:chemistry* / Q000648:ultrastructure; D008954:Models, Biological; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D009032:Mosquito Control / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D015996:Survival Rate; D013912:Threonine / Q000737:chemistry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D013912:Threonine
keywords: Cry toxins; Cyt toxins; ELISA; Hydrogen-bonded stabilization; Receptor-binding subdomain; Synergism