Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin

• Main Authors: Tararat Khaokhiew, Chanan Angsuthanasombat, Chamras Promptmas
• Other Authors: Mahidol University
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Immunology and Microbiology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/27120

Surface-exposed loop residues, Pro389 (β6- β7 loop), Glu417 (β8- β9 loop), Tyr455 and Asn456 (β10-β11 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 (β6-β7/β8- β9 loops), P389A/Y455A, P389A/N456A (β6- β7/β10-β11 loops), E417A/Y455A and E417A/N456A (β8-β9/β10- β11 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. © 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.