Aromatic Residues on the Side Surface of Cry4Ba-Domain II of Bacillus thuringiensis subsp. israelensis Function in Binding to Their Counterpart Residues on the Aedes aegypti Alkaline Phosphatase Receptor

Abstract

Receptor binding is a prerequisite process to exert the mosquitocidal activity of the Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis. The beta-sheet prism (domain II) and beta-sheet sandwich (domain III) of the Cry4Ba toxin have been implicated in receptor binding, albeit the precise binding mechanisms of these remain unclear. In this work, alanine scanning was used to determine the contribution to receptor binding of some aromatic and hydrophobic residues on the surface of domains II and III that are predicted to be responsible for binding to the Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP) receptor. Larvicidal activity assays against A. aegypti larvae revealed that aromatic residues (Trp327 on the β2 strand, Tyr347 on the β3–β4 loop, and Tyr359 on the β4 strand) of domain II were important to the toxicity of the Cry4Ba toxin. Quantitative binding assays using enzyme-linked immunosorbent assay (ELISA) showed similar decreasing trends in binding to the Aa-mALP receptor and in toxicity of the Cry4Ba mutants Trp327Ala, Tyr347Ala, and Tyr359Ala, suggesting that a possible function of these surface-exposed aromatic residues is receptor binding. In addition, binding assays of the Cry4Ba toxin to the mutants of the binding residues Gly513, Ser490, and Phe497 of the Aa-mALP receptor supported the binding function of Trp327, Tyr347, and Tyr359 of the Cry4Ba toxin, respectively. Altogether, our results showed for the first time that aromatic residues on a side surface of the Cry4Ba domain II function in receptor binding. This finding provides greater insight into the possible molecular mechanisms of the Cry4Ba toxin.

Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis uses both domains II and III to bind to its receptor—Aedes aegypti alkaline phosphatase

“…In addition, our results showed that the binding affinity of each domain II or domain III fragment to the Aa-mALP receptor was lower than that of the full-length toxin containing both domains ( K d = 76.19 ± 4.26 nM), implying that the Cry4Ba used both domains II and III to bind to the Aa-mALP receptor. This finding, together with our other works (that reported mutations on some residues on the surface of domains II and III affected binding to the Aa-mALP receptor [ 16 , 17 ]), strengthened the proposition that both domains II and III of the Cry4Ba toxin contributed to binding to the Aa-mALP receptor. In addition, the pulldown assay results revealed binding between the Cry4Ba domains II or III to the Aa-mALP receptor, supporting the interaction between the Aa-mALP receptor and both domains of the Cry4Ba toxin.…”

“…Each receptor binding domain of Cry4Ba was successfully isolated from the full-length toxin and showed interaction with the purified Aa-mALP receptor. Even binding interaction assays were performed in vitro , with the results supporting our previous studies in which amino acid residues on domains II and III were shown to involve receptor binding and hence toxicity toward A. aegypti larvae [ 16 , 17 ]. Both domains of the Cry4Ba toxin seemed to be related in receptor binding to exhibit the larvicidal activity of the toxin.…”

“…In addition, silencing of ALP1 led to tolerance to Cry4Ba and Cry11Aa of A. aegypti larvae [ 13 ]. Amino acid substitution of some residues on the apical loops of domains II and III of the Cry4Ba toxin showed effects on both binding to the ALP receptor and toxicity against A. aegypti larvae [ 13 , 16 , 17 ], which implied that the specific determinant binding to the Aa-mALP receptor relied on both domains II and III. Previously, domain II–III and III fragments, which were cloned from the Cry4Ba toxin, showed binding to midgut tissue sections and the brush border membrane proteins of A .…”

“…israelensis, membrane-bound ALP (Aa-mALP) and APN (Aa-mAPN) located in the Aedes aegypti midgut were reported as its functional receptors [ [12] , [13] , [14] , [15] ]. In our recent studies, we showed that mutation on surface residues on the Cry4Ba domains II and III affected binding to the Aa-mALP receptor and hence toxicity toward A. aegypti larvae [ 16 , 17 ]. In addition, we showed that the isolated domain III fragment was bound to the Aa-mALP receptor with a dissociation constant ( K d ) of about 110 nM [ 17 ].…”

In silico Screening of Plectranthus ampoinicus and Hyptis suaveolens Phytochemicals: Novel Repellents Targeting Odorant Binding Proteins of Aedes aegypti and Aedes albopictus

“…aegypti and Ae. albopictus are responsible for transmitting dengue virus (DENV) (Thammasittirong and Thammasittirong, 2023), chikungunya virus (CHIKV), and yellow fever virus (YFV) on a global scale (Kamgang et al 2019). In South and Southeast Asia, chikungunya virus (CHIKV) is predominantly spread in urban areas through a cycle involving Ae.…”