Mutations at Domain II, Loop 3, of Bacillus thuringiensis CryIAa and CryIAb δ-Endotoxins Suggest Loop 3 Is Involved in Initial Binding to Lepidopteran Midguts

Abstract

Alanine substitutions of loop 3 residues, 438 SGF-SNS 443 , of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly 439 ) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe 440 , is distinctly different from our previous observation that alanine substitution of Phe 371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M. sexta (Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., and Dean, D. H. (1995) J. Bacteriol. 177, 2276 -2282). Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440 AAGA 443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M. sexta (28 times less). The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects. However, alanine substitution of residues 437 LSQ 439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B. mori or M. sexta. These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.

Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

“…It had previously been shown that loop regions of domain II harbor receptor-binding epitopes in several insecticidal Cry toxins (23,39,41,45,53). It has also been shown that some mutations in loop regions do not affect initial receptor binding, as measured by competition binding, but affect irreversible binding, suggesting that they affect insertion of the toxin into the membrane (39,53).…”

“…A mutation in loop 2 of Cry1Ab decreases the toxicity to Manduca sexta by affecting irreversible binding, but the same mutant affects neither toxicity nor binding affinity to Heliothis virescens (37). On the other hand, alanine substitutions of loop 3 residues G439 and F440 in Cry1Ab affect the initial receptor binding and toxicity in both M. sexta and H. virescens (41). These loops are proposed to be exceptional targets for genetic engineering to create more potent toxins with diverse insect specificity (38).…”

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

“…Binding of Cry1Ab toxin with Bt-R 1 facilitates removal of helix ␣-1, triggering toxin oligomerization (2). The oligomer gains binding affinity to the second receptor, APN, and this interaction enhances insertion of the oligomer into the membrane (3,40). We previously hypothesized that sequential interaction of the Cry1Ab toxin with the two receptors involves structural changes of binding epitopes upon oligomerization (16).…”

“…Published data regarding the role of this binding region in the interaction with both receptor molecules are incomplete because binding of loop 3 mutant oligomers with the two receptor molecules was not previously analyzed. Mutagenesis studies of Cry1Ab and Cry1Ac loop 3 previously performed indicated that this amino acid region was important for binding with M. sexta BBMV and toxicity (40,41). In addition, alanine substitutions of loop 3 residues in Cry1Ab and Cry1Ac toxins showed a correlative effect on APN binding, suggesting that their effects on toxicity were due to defects in this binding (22,23).…”

Location of theBombyx moriAminopeptidase N Type 1 Binding Site onBacillus thuringiensisCry1Aa Toxin

“…Domain I is involved in membrane insertion and pore formation (5,21,25,43). Domain II plays a role in binding to the insect brush border membranes (16,39,42,44,46,61,62,63,64,72,76) or receptor molecules (23). Domain III is involved in several functions, including determination of insect specificity (8,9,10,38,39,49), recognition of N-acetylgalactosamine (GalNAc) on receptor molecules by a lectin-like structure (4,30,31,36,40), proteolytic protection (43), and ion channel conductivity (6,69,75).…”