Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of theBacillus thuringiensisCry4Ba 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.

Cadherin, Alkaline Phosphatase, and Aminopeptidase N as Receptors of Cry11Ba Toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti

“…For Cry4Ba, it has been revealed that loops ␤6-␤7, ␤8-␤9, and ␤10-␤11 play a role in Cry4Ba toxicity, and combinations of two-loop mutants (loops ␤6-␤7/␤8-␤9, loops ␤6-␤7/␤10-␤11, and loops ␤8-␤9/␤10-␤11) exhibited reduced binding to apical microvilli of the Ae. aegypti larval midgut (28,41). However, there are no data on whether other loops of the Cry4Ba toxin are involved in toxicity and binding activity.…”

Mosquito Resistance to Bacterial Larvicidal Toxins

“…The central helix is in fact not entirely hydrophobic, but rather exhibits an amphipathic character, as all of its polar or charged side-chains in the interhelical space are engaged in hydrogen bonds or salt bridges (Grochulski JMB 1995) [15] (Li Nature 1991) [17] (Boonserm J Bacteriol 2006) [19] (Boonserm JMB 2005) [20]. This is also the case for all the outer helices which are oriented with their polar or charged residues forming the outer surface of the helical bundle (Grochulski JMB 1995) [15] (Li Nature 1991) [ [27] have also been shown to be involved in receptor binding. The C-terminal domain ( 12-23) consists of two twisted anti-parallel -sheets that are arranged in a jelly-rolllike topology or a face-to-face sandwich.…”

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

“…Domain II is a β-prism of three anti-parallel β-sheets packed around a hydrophobic core with exposed loop regions, and domain III is a β-sandwich of two anti-parallel β-sheets; both domains are involved in midgut protein recognition (Bravo et al, 2007). In the case of Cry4Ba toxin, mutagenesis studies have shown that domain II loop 2 region is important for A. aegypti toxicity (Khaokhiew et al, 2009). …”