Mode of action of Cry2Aa, a Bacillus thuringiensis dual active insecticidal crystal protein /

Cry2Aa, a Bacillus thuringiensis 5-endotoxin, is specific to both dipteran and lepidopteran insects. Upon digestion by gypsy moth {Lymantria dispar) midgut proteases, the first 49 amino acids at the N-terminus are removed from the 63 kDa protoxin, to form a 58-kDa active fragment. The protease-resistant core is comprised of ValSO as the first amino acid, which is located at the loop between the aO and a1 helices of domain I. The toxin’s potency was lost when further in vitro midgut protease processing continued. The loss in toxicity was caused by proteolytic cleavage at the carboxyl end of Leu 144, which is on the loop between aS and a4 of domain I. To prevent the production of the non-toxic fragment, five mutant proteins, L144D, L144A, L144G, L144H, and LI44V, were constructed. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. However, the mutant proteins were as toxic to the insects as the wild type protein, indicating that the cleaved fragment is associated with the rest of the entire protein molecule in the midgut environment. The cleavage was also protected when the toxin is bound to the midgut membrane. The role of the 49-amino acid length of the N-termina! sequence of Cry2Aa was investigated. This short peptide sequence contains one extra a helix, called aO, which is located between Phe24 and His46. Gene deletion and site-directed mutagenesis results indicated that the presence of this a helix is necessary for the formation of biological active inclusion of Cry2Aa. Deletion of the cry2Aa gene at the aO helix, and deletion of the entire N-terminus region led to production of the non-soluble inclusion bodies that were non-toxic to both L. dispar and A. quadrimacuiatus. However, expression cry2Aa gene that contains the aO helix, but lacking the Val3 Asn17 region, gave the soluble, and toxic crystal inclusions. Tertiary structure of Cry2Aa reveals a number of intramolecular and intermolecular interactions contributed by the amino residues in aO. The intramolecular interactions might stabilize, and facilitate folding of the Cry2Aa protoxin, as demonstrated by the observation that the melting temperature of the Cry2Aa protoxin and the mutant protein containing aO were 7 °C higher than the active Cry2Aa fragment. Mutation of amino acids involved in intramolecular interactions lowered the yield of the inclusion bodies. Loss in the inclusion amount was also found when the residues involved in intermolecular bondings were mutated. Finally, results from intensive site-directed mutagenesis of amino acids on loop 1 and loop 2 of domain II and bioassays against A. quadrimacuiatus and L dispar revealed that specificity of Cry2Aa to dipteran insects was conferred by amino acids in loop 1, while the amino residues on loop 2 are responsible for Lepidoptera specificity. The mutant proteins A2 (F320-321AAA), F320A, and P321A were less toxic to A. quadrimacuiatus larvae. Change in toxicity was related to change in competition binding affinity. However, the same mutant proteins were as toxic to L dispar larvae as the wild type protein and change in binding properties to L. d/spar BBMV was not observed. In contrast, the mutant proteins obtained from the alterations in the region, i. e. , A5 (DRE383-5AAA), D383V, R384A, and E385A, were less toxic to only L dispar. Results from dissociation experiments indicated that these mutant proteins were not involved in irreversible binding. The dramatic changes found in these mutant proteins were reversible binding. Therefore, Initial binding of amino acids on loop 1 and loop 2 of Cry2Aa might be a factor that determines the toxin dual specificity. The obtained results concerning active fragment processing, solubility, and specificity of Cry2Aa might reveal a fundamental mode of action of Cry2Aa and other Cry toxins from Bacillus thuringiensis. Active fragments of Cry toxins seem to contain all seven a-helices to retain their biological activity. The results also indicated that formation of the bioactive crystal in the bacterial cells requires not only the active fragment portion, but also the extra-portion of the protoxins. These peptide portions might facilitate both protein folding and assembly of the protoxin molecules. Finally, the results obtained from the Cry2Aa-receptor binding and toxin specificity experiments might help in engineering more potent and more specific Cry toxins, which will be used to control the certain target insects. iv Dedicated to my father