title: Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin.
abstract: Surface-exposed loop residues, Pro(389) (beta(6)-beta(7) loop), Glu(417) (beta(8)-beta(9) loop), Tyr(455) and Asn(456) (beta(10)-beta(11) loop), in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin have been previously demonstrated to be crucial for toxicity. Herein, five combinations of two-loop mutants, P389A/E417A (beta(6)-beta(7)/beta(8)-beta(9) loops), P389A/Y455A, P389A/N456A (beta(6)-beta(7)/beta(10)-beta(11) loops), E417A/Y455A and E417A/N456A (beta(8)-beta(9)/beta(10)-beta(11) loops), were constructed as a means of examining a correlative effect of these three critical loops on Cry4Ba toxicity. All 130-kDa mutant protoxins were overexpressed as inclusion bodies in Escherichia coli with yields comparable to the wild-type toxin. In addition, all mutant toxins were structurally stable upon solubilization and trypsin activation in carbonate buffer, pH 9.0. Interestingly, E. coli cells expressing all the double-loop mutants showed an almost complete loss in toxicity against Aedes aegypti mosquito larvae while their corresponding single-loop mutants exhibited reduced activity of approximately 50%. Moreover, in situ binding analysis revealed that the 65-kDa purified toxins representing each pairwise mutant exhibited reduced binding to apical microvilli of A. aegypti larval midgut when compared with the single mutants. Altogether, the data demonstrate for the first time that all these three surface-exposed loops of the Cry4Ba toxin are equally involved in receptor binding and hence toxicity.
authors.affiliation: Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok-Noi, Bangkok, Thailand.
authors.forename: Tararat
authors.initials: T
authors.lastname: Khaokhiew
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
authors.forename: Chamras
authors.initials: C
authors.lastname: Promptmas
mesh_terms: D000330:Aedes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D019476:Insect Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis