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dc.contributor.authorAnon Thammasittirongen_US
dc.contributor.authorSutticha Na Ranong Thammasittirongen_US
dc.contributor.authorChompounoot Imtongen_US
dc.contributor.authorSathapat Charoenjotivadhanakulen_US
dc.contributor.authorSomsri Sakdeeen_US
dc.contributor.authorHui Chun Lien_US
dc.contributor.authorSiriporn Okonogien_US
dc.contributor.authorChanan Angsuthanasombaten_US
dc.description.abstractIn addition to the receptor‐binding domain (DII), the C‐terminal domain (DIII) of threedomain Cry insecticidal δ‐endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21kDa high‐purity isolated DIII fragment derived from the Cry4Ba mosquito‐specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor—Aedes aegypti membrane‐bound alkaline phosphatase (Aa‐mALP). Binding analysis via dotblotting revealed that the Cry4Ba‐DIII truncate was capable of specific binding to nitrocellulosebound Aa‐mALP, with a binding signal comparable to its 65kDa Cry4Ba‐R203Q full‐length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba‐DIII exhibited a rather weak binding to Aa‐mALP with a dissociation constant (Kd) of ≈1.1 ×10−7 M as compared with the full‐length toxin. Intermolecular docking between the Cry4Ba‐R203Q active toxin and AamALP suggested that four Cry4Ba‐DIII residues, i.e., Glu522, Asn552, Asn576, and Leu615, are potentially involved in such toxin–receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A‐impaired toxin also exhibited a reduction in binding capability to the surface‐immobilized Aa‐mALP receptor, while two bio‐inactive DII‐mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C‐terminal domain of Cry4Ba for serving as a potential receptor‐binding moiety in which DIII‐Leu615 could conceivably be exploited for the binding to Aa‐mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.en_US
dc.subjectEnvironmental Scienceen_US
dc.subjectPharmacology, Toxicology and Pharmaceuticsen_US
dc.titleBacillus thuringiensis cry4ba insecticidal toxinexploits leu<sup>615</sup> in its c‐terminal domain to interact with a target receptor—aedes aegypti membrane‐bound alkaline phosphataseen_US
article.volume13en_US University, Kamphaeng Saen Campusen_US Chi Universityen_US of Molecular Biosciences, Mahidol Universityen_US of Songkla Universityen_US Mai Universityen_US Institute for Research and Development (BIRD)en_US
Appears in Collections:CMUL: Journal Articles

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