1887

Abstract

Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the insecticidal CrylAc δ-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1, 2 and 3, respectively) in domain II of the δ-endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop 1), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed δ-endotoxin protein at wild-type levels which was stable upon activation by gut extract or trypsin. Toxicity assays for all the fully stable mutants using larvae showed that G312, Y367, R368, R369, S438 and G439 are important for activity. Wild-type toxin was then labelled with [S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues G312 and S438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.

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1996-07-01
2021-09-25
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