Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-Terminal Domain to Interact with a Target Receptor—Aedes aegypti Membrane-Bound Alkaline Phosphatase

Abstract

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal d-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 nitrocellulose-bound 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 Aa-mALP 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.

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

“…The docking result showed that the major domain II residues that function in binding to the Aa-mALP receptor are on a side surface rather than only as residues on the beta-hairpin loops located on the underside of the toxin. In our previous work, some binding residues from this docking result were studied and we found that Leu 615 in the β22–β23 loop of the Cry4Ba domain III toxin was a crucial residue for Aa-mALP receptor binding [ 21 ]. In the present study, aromatic and hydrophobic residues of Cry4Ba domain II (Trp 327 , Tyr 347 , Ile 358 , and Tyr 359 ) and domain III (Phe 490 and Leu 517 ) and their counterpart interacting residues (Gly 513 , Ser 490 , His 500, Phe 497 , Leu 53 , and Leu 52 , respectively) on Aa-mALP ( Figure 1 ) predicted as responsible for binding were selected to study their relevance to binding and toxicity.…”

“…For the Cry4Ba toxin, several amino acid residues on β-hairpin loops, especially those located on the lower part of domain II such as Thr 386 , Ser 387 , Ser 388 , Pro 389 , Ser 390 , and Asn 391 on the β6–β7 loop, Glu 417 on the β8–β9 loop, and Tyr 455 and Asn 456 on the β10–β11 loop, have been reported to be important for binding and toxicity [ 16 , 22 ]. Our previous study [ 21 ], using molecular docking, revealed a possible Cry4Ba toxin—Aa-mALP receptor interaction in which surface residues on the side of the domain II, such as residues on β2 and β4 and residues on the β-hairpin loops of domains II and III rather than only the residues on the β-hairpin loops at the bottom of domain II, are responsible for binding to the Aa-mALP receptor ( Figure 1 ). According to a previous study, three fundamental hotspot residues (tryptophane, tyrosine, and arginine) were reported to contribute more significantly to binding affinity in protein–protein interfaces compared with others (for review, see [ 27 ]).…”

“…A number of A. aegypti midgut proteins have been reported to interact with the Cry4Ba toxin [ 15 ]; among these, glycosylphosphatidylinositol (GPI)-anchor alkaline phosphatase (ALP) and aminopeptidase N (APN) have been well characterized as receptors for the Cry4Ba toxin [ 16 , 17 , 18 , 19 , 20 ]. In our recent study, we provided evidence that both the full-length Cry4Ba toxin and its isolated domain III could bind to the membrane-bound ALP (Aa-mALP) receptor from A. aegypti larvae [ 21 ].…”

“…Mutation of these residues affected the binding of Cry4Ba to A. aegypti midgut brush border membrane vesicles (BBMV) [ 16 , 22 ] or the purified ALP1 isoform receptor [ 16 ]. In a recent study using molecular docking interaction and site-specific mutagenesis, we have shown that Leu 615 on the β22–β23 loop of Cry4Ba-DIII was involved in binding to the purified Aa-mALP receptor and related to toxicity against A. aegypti larvae [ 21 ]. However, other residues of the Cry4Ba domains DII and III predicted by a molecular docking study as binding residues, especially aromatic and hydrophobic side chains, remain to be explored.…”

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

“…While various studies have suggested that the C-terminal domain—DIII could be implicated in preserving structural integrity or in determining target insect specificity [ 27 , 28 , 29 , 30 , 31 , 32 , 33 ], we have recently revealed that Cry4Ba-DIII could function as a tight-binding anchor for lipid membrane bilayers, highlighting its potential contribution to toxin-membrane interactions to mediate toxin activity [ 34 ]. More recently, we have demonstrated that Cry4Ba-DIII could also act as a receptor-binding moiety, signifying another contribution to toxin interactions with its target protein receptor— Aa -mALP in mediating larval toxicity [ 35 ].…”