Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.Bacillus thuringiensis subsp. israelensis has received considerable attention for mosquito control because of its specific and potent toxicity (15). B. thuringiensis subsp. israelensis-based microbial insecticides have been widely used as active components for integrated management of mosquitoes (11, 13, 33, 34). B. thuringiensis subsp. israelensis produces at least four major crystal toxins (Cry toxins), namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (5). Cry4Aa exhibits specific toxicity against Anopheles, Aedes, and Culex mosquito larvae (15, 27). The 130-kDa Cry4Aa protoxin is released from the protein crystal upon ingestion by susceptible mosquito larvae and is activated by gut proteases into two protease-resistant fragments with molecular masses of 20 and 45 kDa through intramolecular cleavage of a 60-kDa intermediate (39). The three-dimensional structure of Cry4Aa has been determined by X-ray crystallography at a resolution of 2.8 Å (6). The structure of Cry4Aa is similar to the structures of previously characterized Cry toxins (24, 26, 31) that are composed of three domains (domains I, II, and III). In general, domain I, which is located in the N-terminal region, is composed of seven amphipathic α-helices and is thought to participate in membrane insertion. Domain II, which consists of three antiparallel β-sheets, is a putative receptor binding domain (Fig. ​(Fig.1).1). In particular, the loops in domain II that are exposed on the surface of the toxin molecule vary significantly in length and amino acid sequence among Cry toxins (31) and are thought to be receptor binding sites. Domain III in the C-terminal region contains two antiparallel β-sheets that form a β-sandwich fold with a jellyroll topology (31). Domain III is assumed to be involved in structural integrity, membrane protein recognition, or both (23, 24, 30).Open in a separate windowFIG. 1.Three-dimensional structure of Cry4Aa domain II. The structure was generated with PyMOL software (8) using the Cry4Aa PDB code (6). The amino acid sequences and corresponding regions of loops 1, 2, and 3 are indicated by blue, red, and yellow, respectively. The amino acid sequences of β-strands adjacent to the loops are underlined.The insecticidal mechanism of Cry toxin involves multiple steps, including ingestion by susceptible insects, solubilization in the alkaline midgut juice, activation by trypsin-like midgut proteases, binding to specific receptors on midgut epithelial cells, and then insertion into the plasma membrane followed by the formation of cation-selective channels or pores (26, 31, 34, 41). According to the colloid-osmotic lysis model, these channels or pores allow ions and water to pass into the cells, resulting in destruction of the membrane potential, cell swelling, cell lysis, and eventual death of the host (20, 21). Thus, the mechanism seems to be very complicated and is affected by multiple factors. The binding of the toxin to the specific receptor is considered a vital step for specific insecticidal activity (35). In fact, modification of the receptor molecules has been reported for insects resistant to certain Cry toxins (12, 22, 36).In a search for the functional structures of Cry4Aa, we previously constructed various loop replacement mutants with mutations in the three major loops in domain II and showed that the replacement of loop 2 resulted in a significant loss of mosquitocidal activity. Replacement of loops 1 and 3 of Cry4Aa also affected mosquitocidal activity, but it did not eliminate it (17). In this study, to further characterize the loops, we constructed Cry4Aa mutants in which individual amino acids in the loops were replaced with alanine and analyzed the mutants to determine their mosquitocidal activity against Culex pipiens larvae. We also analyzed the structural integrity of the Cry4Aa mutant proteins subjected to proteolytic digestion and their binding affinity to brush border membrane vesicles (BBMV) prepared from C. pipiens larvae.