Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae.

The three-domain Cry4Aa toxin produced from subsp. was previously shown to be much more toxic to mosquito larvae than its closely related toxin-Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from larvae, mALP1263and mALP1264, predicted to be GPI-linked was cloned and functionally expressed in (9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both mALP isoforms share significant sequence similarity to -mALP-a Cry4Ba toxin receptor. In cytotoxicity assays, 9 cells expressing mALP1264, but not mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each mALP expressed on the cell membrane surface. Molecular docking of the -mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to -mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain-DII, including Thr, Tyr and Lys in the β10-β11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain-DIII to interact with such a counterpart receptor. Ala-substitutions of selected β10-β11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against larvae. Further substitution of Lys with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔG) in Cry4Aa--mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔG value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same membrane-bound ALP isoform-mALP1264 for mediating differential toxicity against target larvae.