**2. Results**

#### *2.1. Interactions of Destruxin A with Three Proteins by BLI and CETSA Analysis*

In order to evaluate whether BmLamin-C, BmArgRS and BmPRP1 proteins interact with DA, we conducted the experiments of bio-layer interferometry (BLI) in vitro and cellular thermal shift assay (CETSA) in vivo. In BLI assays, the results indicated that the responses of BmArgRS and BmLamin-C proteins were positively correlated with DA concentrations. There were affinity constant (KD) values of 5.53 × 10−<sup>5</sup> and 8.64 × 10−<sup>5</sup> M respectively for DA with BmArgRS and BmLamin-C (Table 1, Figure 1B). However, the results showed that BmLamin-C is not a DA-binding protein, because a significant correlation between DA concentration and response of BmPRP1 was not detected. Meanwhile, the results of CETSA experiments exhibited that DA induced the thermal stability shift of BmArgRS and BmLamin-C but not BmPRP1 (Figure 1C), which suggested that the two proteins interact with DA in vivo. Obviously, both the BLI and CETSA experiments provided in vitro and in vivo evidences for that DA binds to BmArgRS and BmLamin-C but not BmPRP1.


**Table 1.** Detailed results of bio-layer interferometry (BLI) assay.

1Kon: association rate constant; 2 Kdis: dissociation rate constant; 3 KD: affinity constant.

#### *2.2. Key Sites of Interaction of DA with BmArgRS and BmLamin-C*

In the circular dichroism (CD) tests, the scanning results in 190–260 nm ultraviolet region indicated the CD shifts of DA-treated BmArgRS and BmLamin-C, which sugges<sup>t</sup> that DA damages the α-helixes of the two proteins (Figure 2A). Meanwhile, in the 250–340 nm ultraviolet scanning, the protein CD changes caused by DA were found, which indicated that DA brings on the transformation of disulfide bonds and some side chains of BmArgRS and BmLamin-C (Figure 2A). These data provide the evidences for the interactions of DA with BmArgRS and BmLamin-C.

Furthermore, in analysis of homologue modeling (Figure 2B,C), DA interacts with BmArgRS and BmLamin-C with the scores −9.71 and −8.08 kcal/mol, respectively. The molecular docking predicted that there are many hydrogen bonds between DA and BmArgRS, which provide a pocket consisted of Lys228, His231, Gln437, Lys475, Val468, and Asp434 to DA binding (Figure 2D). However, there is only a hydrogen bond between DA and Lys528 in BmLamin-C (Figure 2E), although a pocket formed by His524, Thr526, Lys528, Glu530, Ser535, Ile552, and Met554 was predicted.

Based on the analysis above, each of amino acids of comprising hydrogen bond was mutated to alanine and the interactions of all mutant proteins with DA were investigated through BLI assay. Interestingly, in BmArgRS, 4 mutants, His231Ala, Lys228Ala, Asp434Ala, and Gln437Ala, displayed no interaction with DA (Figure 3). However, the interactions were still found in the mutants Val468Ala and Lys475Ala with DA, because the KD values of 5.3 × 10−<sup>5</sup> and 5.71 × 10−<sup>6</sup> were respectively recorded, which were at same level compared with wild type (Figure 3). Those illustrated that DA binds to BmArgRS in the pocket including Lys228, His231, Asp434, and Gln437. Strikingly, the interaction site is just located in the conserved enzyme active catalysis domain of BmArgRS. Likewise, the His524Ala and Lys528Ala mutants of BmLamin-C exhibited no interactions with DA, which implied that Lamin tail domain is the DA-binding site (Figure 3).

**Figure 2.** Structural evidences of interaction between DA with BmArgRS and BmLamin-C. (**A**) Profiles of circular dichroism tests indicating the effects of DA on proteins secondary structures. **A1**, **A2**, 190–260 nm and 250–340 nm ultraviolet region of BmArgRS interact with DA respectively. **A3**, **A4**, 190–260 nm and 250–340 nm ultraviolet region of BmLamin-C interact with DA respectively. (**B**,**C**) Homologous modeling of BmArgRS and BmLamin-C respectively. (**D**,**E**) Binding pose of DA with BmArgRS and BmLamin-C respectively.

**Figure 3.** Key amino acid sites for interaction of DA with BmArgRS and BmLamin-C by BLI tests. (**A**) The interactions of DA with the mutants of BmArgRS and BmLamin-C. (**B**,**C**) Sketch of the domains of BmArgRS and BmLamin-C and key amino acid sites for DA binding.

#### *2.3. Gene Expression Levels of Three Proteins in Bm12 Cells*

We investigated DA dosage- and time-depend affecting expression three genes in Bm12 cells. The results indicated that there were no obvious relations between genes expression levels of three proteins in DA dosage- and time-depend manner. Totally, these genes had up-regulation of <2 folds, only BmArgRS was up-regulated by 3-fold in relative high dosage 200 μg/mL at 6 h post-treatment (Figure 4A). It is suggested that DA only leads to mild changes in gene expression levels of the three proteins.

**Figure 4.** Effects of DA on BmArgRS and BmLamin-C of Bm12 cell. (**A**) Analysis of gene expression under DA stress by qPCR test. (**B**) Cytotoxicity of DA against Bm12 cells by RNAi treatments. (**C**) Enzyme activity of BmArgRS inhibited by DA.

#### *2.4. Changes of DA Cytotoxicity and BmArgRS Enzyme Activity in Bm12 Cells*

The results indicated that the toxicities of DA against Bm12 cells were decreased by more than 50% after the genes of *BmArgRS* and *BmLamin-C* were knocked down (Figure 4B). In addition, the enzyme activity of BmArgRS was decreased under DA exposure, because a negative correlation between enzyme activity and DA dosage was found (Figure 4C). This might explain that DA binds to enzyme active center of BmArgRS so as to inhibit its catalytic function.
