Mutations at Cys70, Cys91, Cys183, and Cys258 did not affect crystal formation except for protein production of Cry49Aa
The cysteine residues played an important role in participating in toxin formation, toxicity and interaction (Girard et al., 2008; Promdonkoy et al., 2008; Boonyos et al., 2010). Here, C70, C91, C183 and C258 in Cry49Aa were changed to alanine. Cry49Aa wild type and mutants were constructed and expressed in B. thuringiensis BMB171. All mutant proteins were produced small spore-associated crystals that showed a bipyramidal morphology as the wild type. While, the mutant proteins showed comparable yield to that of the wild type except that the C91A mutant was low (Fig. 1A). These results clearly showed that replacements at Cys70, Cys91, Cys183, and Cys258 by alanine did not affect crystal formation and morphology of Cry49Aa.
Disulfide bonds are key characterizing structural and functional properties (Girard et al., 2008; Promdonkoy et al., 2008; Boonyos et al., 2010). The SDS-PAGE with and without a reducing agent (10 mm β-Mercaptoethanol) was used to analyze whether disulfide bond could be formed between cysteine residues at position 70, 91, 183, and 258 in Cry49Aa. The results showed that there was no difference in the mobility of the major bands formed as Cry49Aa monomers from the wild type and mutants with and without reducing agent, while, a weak dimer band was observed in the samples without reducing agent (Fig. 1A, B). These results demonstrated that Cys70, Cys91, Cys183, and Cys258 may not participate in the intra- or intermolecular disulfide bonds formation in Cry49Aa molecules. Amino acid sequence analysis showed there are 2 cysteine residues (Cys479, Cys562) in N‑terminal domain of active Cry48Aa toxin is responsible for Cry48Aa–Cry49Aa interaction (Guo et al., 2016, 2020). Thus, it is supposed that one or more cysteine residues in Cry48Aa could result in disulfide bond formation with Cys70, Cys91, Cys183, and Cys258 in Cry49Aa.
Cys91, Cys183, and Cys258 in Cry49Aa are crucial for the mosquito-larvicidal activity
The mosquito-larvicidal activity of the Cry49Aa mutants against C. quinquefasciatus larvae was tested by mixing with equimolar amounts of Cry48Aa toxin from partially purified crystals. The C183A mutant and C258A mutant completely lost their total biological activity, while the C91A mutant significantly reduced the toxicity. However, the alanine substitution at Cys70 showed a comparable toxicity to that of the wild type (Table 2). These results revealed that the cysteine at the position 91, 183, and 258 in Cry49Aa is absolutely crucial for toxicity of the Cry49Aa/ Cry48Aa toxin, whereas Cys70 is less important. The cysteine replacement with alanine of Cry49Aa toxin did not affect the crystal formation and structure described as above, it is possible to affect hydrophobicity or polarity of these regions.
Table 2 Mosquito larvicidal activity of Cry49Aa wild type and mutants against Culex quinquefasciatus larvae
Cry48Aa +
|
LC50 (ng/mL)
|
Cry49Aa (wild type)
|
7.3(5.1-9.8)
|
Cry49Aa-C70A
|
6.8(5.1-8.9)
|
Cry49Aa-C91A
|
45.6(34.1-68.5)
|
Cry49Aa-C183A
|
inactive
|
Cry49Aa-C258A
|
inactive
|
Effect of cysteine substitutions on Cry48Aa- Cry49Aa interaction
Cry49Aa subunit plays a crucial role in the action mechanism of Cry48Aa/ Cry49Aa two-component toxins, while Cry48Aa-Cry49Aa interaction is one of the key steps in achieving their toxic activity against larvae (Jones et al., 2007; Guo et al., 2016, 2020). In our study, the far-Western dot blot analysis was used to explore the possible role of Cry49Aa cysteine in the intermolecular Cry49Aa- Cry48Aa interaction. The results showed the C70A mutant, interacted with Cry48Aa, displayed the strongest signal with comparable sensitivity to that of the wild type. The C91A and C258A mutants had moderately lower signals than that of the wild type, while the C183A mutant showed the considerably weakest signal compared to the wild type (Fig. 2). Thus, the Cys91 Cys183, and Cys258 located in the Cry49Aa N-terminus may be required for intermolecular Cry48Aa-Cry49Aa interaction. These results suggested that the weaker Cry49Aa- Cry48Aa interaction caused by the Cry49Aa N-terminal mutations at C91, C183 and C258 may contribute to the decrease or loss of toxicity of the toxin.
Effect of cysteine mutations on binding to C. quinquefasciatus BBMFs
The toxin interacted with receptor proteins present in insect larvae midgut cells, leading to membrane insertion and pore formation, then destroying the cells and killing the larvae (Bravo et al., 2007; Jones et al., 2008; de Melo et al., 2009). Previous studies had demonstrated that Cry49Aa had high binding capacity to C. quinquefasciatus BBMFs (Guo et al., 2016; Rezende et al., 2017). Here, the affinity of Cry49Aa mutants bound to BBMFs was determined by using 10 nM labelled Cry49Aa wild type toxin incubated with increasing concentrations of unlabelled mutant toxins. The results showed that the wild type Cry49Aa component had high binding capacity to C. quinquefasciatus midgut BBMFs with a half-maximal inhibitory concentration (IC50) of 24.3± 5.7 nM. While, the C70A and C91A mutants had comparatively higher competitive binding capacities to BBMFs than that of the wild type with IC50 of 9.5± 2.1 and 13.9± 3.2 nM, respectively. Whereas, the C183A and C258A mutants had poorer binding affinities for BBMFs than that of the wild type with IC50 of 154.6± 20.3 and 186.2± 25.3 nM, respectively (Fig. 3). These results imply that mutations at C183 and C258 result in weaker interaction between toxin and its receptors which may contribute to loss of toxicity. Previous investigation suggesting that Cry49Aa C-terminal fragment located between S349 and N464 is essential and sufficient for receptor binding, and the N-terminal region is important for interacting to Cry48Aa (Guo et al., 2016). Although C183 and C258 are in the N-terminal region, but closer to the C-terminus, both residues may locate in a close proximity to the C-terminal part after protein folded into the functional 3D structure, leading to weaken the receptor binding ability and lose its toxicity. In addition, the C91A mutant has higher receptor binding capacity but weaker ability to form Cry48Aa-Cry49Aa complex, which may result in decreasing the virulence. Thus, toxin oligomerization was also a critical step in the mosquito- larvicidal activity, and other factors may also involve in the action mechanism of Cry49Aa/ Cry48Aa toxin.
In general, the replacements at Cys91, Cys183, and Cys258 by alanine did not affect crystal formation but significantly decreased its toxicity and reduced receptor binding and oligomer formation. These cysteine residues may not participate in disulfide bond formation in Cry49Aa, but may affect crystal 3D structures, and then play an important role on Cry48Aa-Cry49Aa complex formation and toxin-receptor interaction. The Cry49Aa, as accessory protein, only combined with Cry48Aa could achieved the insecticidal activity (Jones et al., 2007, 2008; Guo et al., 2016, 2020). However, it is uncertain that mutations at these positions lead to weaken interaction between Cry49Aa mutants and Cry48Aa or BBMFs could account for the loss of virulence. Cys183 and Cys258 in Cry49Aa may play a critical role on conformational change, receptor binding, and/or membrane insertion, because mutations at both positions had more adverse effect than at Cys91. These results provided for useful information to explore the mechanism of action of L. sphaericus Cry48Aa/Cry49Aa toxins.