Construction of Cry2Ab mutants sited-directed on helices α4-α5
Our previous studies demonstrated that helices α4-α5 in Domain I was involved in oligomerization of Cry2Ab since some Cry2Ab mutants (TM152153AA, LF156157AA, NR159160AA, LH183184AA, FI187188AA) failed to assemble 250 kDa oligomers (Fig 1A) (Xu et al., 2018). Those results suggested that the active regions for Cry2Ab oligomerization were limited to V150-R160 in helix α-4 and N182-D190 in helix α-5 (Fig. 1B). To further authenticate single residues for Cry2Ab oligomerization, 11 Cry2Ab mutants site-directed on helix α-4 (V150A, N151A, T152A, M153A, Q154A, Q155A, L156A, F157A, L158A, N159A, R160A) and 9 Cry2Ab mutants site-directed on helix α-5（N182A, L183A, H184A, L185A, S186A, F187A, I188A, R189A, D190A）were constructed using Escherichia coli expression system.
The upstream and downstream DNA fragments of Cry2Ab mutants contained mutation sites were amplified by PCR (Fig. S1A and S1B) and the full-length of DNA fragments of Cry2Ab mutants were obtained by overlap extension PCR (Fig. S1C). The Cry2Ab DNA was ligated to plasmid pET30a and transformation into BL21 competent cells. The recombinant plasmids were verified by PCR using T7 primers and a DNA product about 2300 bp could be amplified (Fig. S2). The recombinant plasmids were further digested with Bgl II and EcoR I and a pET30a fragment (about 5500 bp) and a Cry2Ab fragment (about 1900 bp) could be detected (Fig. S3). Those results suggested that the Cry2Ab DNA was ligated into pET30a. DNA sequencing revealed that the specific sites of 20 Cry2Ab mutants had been replaced by alanine.
Production and identification of Cry2Ab mutants
The expression of Cry2Ab mutants were performed under the induction of 0.5mM isopropyl-β-d-thiogalactoside. SDS-PAGE revealed that all Cry2Ab mutants could be induced-produced with a molecular weight of 65 kDa (Fig. 2A). Those Cry2Ab mutants further purified by a Ni-lDA prepacked column (Fig. 2B) and could be detected by an anti-Cry2Ab antibody (Fig. 2C). Furthermore, proteolysis assay indicated that all Cry2Ab variants could be processed into 50 kDa activated-toxins by PxMJ, which were similar to wild-type Cry2Ab (Fig. 2D). These results revealed that mutations in the helices α4-α5 did not cause a major structural disturbance in Cry2Ab.
Authentication of key residues for Cry2Ab oligomerization
The assembly of 250 kDa oligomer of variants Cry2Ab were evaluated by 8% SDS-PAGE (Fig. 3A). Wild-type Cry2Ab could form 250 kDa oligomers which was similar to our previous reports (Xu et al. 2018). Six Cry2Ab mutants (N151A, T152A, F157A in helix α-4 and L183A, L185A, I188A in helix α-5) failed to assemble 250 kDa oligomers (defined as non-oligomerization group). Eight Cry2Ab mutants (M153A, Q154A, L156A, N159A, R160A in helix α-4 and N182A, H184A, R189A in helix α-5) greatly reduced the assembly of 250 kDa oligomers (defined as reduced oligomerization group). The remaining Cry2Ab variants (V150A, Q155A, L158A in helix α-4 and S186A, F187A, D190A in helix α-5) could aggregate and form 250 kDa pre-pore structure (defined as normal oligomerization group) which were similar to wild-type Cry2Ab.
To further assess the oligomers formation of Cry2Ab, we employed Image J software to evaluate the percentage of oligomer (oligomer / monomer × 100%). As shown in Fig 3B, the proportion of oligomer and monomer in normal oligomerization group was about 80%-110%, which was similar to wild-type Cry2Ab (about 95%). However, this value in reduced oligomerization group was about 40-60% and in non-aggregation group were less than 20%. Those results suggested that residues N151, T152, F157, L183, L185, I188 might serve as key residues for Cry2Ab oligomerization.
Oligomerization is associated with insecticidal activity of Cry2Ab
We further assessed the insecticidal activities of non-oligomerization group (N151A, T152A, F157A, L183A, L185A and I188A) Cry2Ab mutants against second instar larvae of P. xylostella, with wild-type Cry2Ab, V150A and S186A (two Cry2Ab mutants in normal oligomerization group) as positive control (Figure 4). The LC50 values of V150A and S186A was 1.978 and 1.432 μg/cm2, which were close to that of wild-type Cry2Ab (1.458 μg/cm2). However, the LC50 values of N151A, T152A, F157A, L183A, L185A and I188A were 5.097, 4.232, 3.234, 3.083, 3.579 and 3.545 μg/cm2, respectively, all of which were higher than that of wild-type Cry2Ab (1.458 μg/cm2).
Oligomerization is associated with pore-forming activity of Cry2Ab
The pore-forming activities of non-oligomerization group (N151A, T152A, F157A, L183A, L185A and I188A) Cry2Ab mutants were further evaluated by time course of liposome leakage assay. As shown in Figure 5, wild-type Cry2Ab could make pore on liposome and led to high leakage of calcein into solution, which was the same with V150A and S186A. The maximum calcein release percentage caused by wild-type Cry2Ab, V150A and S186A were 60.95%, 55.87%, and 58.73%. However, the leakage of calcein caused by N151A, T152A, F157A, L183A, L185A and I188A were much lower than wild-type Cry2Ab, their maximum calcein release percentage were 24.33%, 20.72%, 35.98%, 32.36%, 37.53% and 32.53%. Those data indicated that non-oligomerization mutants may weaken the pore-forming activities on liposome. Taken together, our data strongly suggested that oligomerization was tightly linked with the pore-forming activity of Cry2Ab.