## 3 Results
### Binding Mode of the (-)-Spirobrassinin with the PSBD1  
The hydrophobic and hydrophilic surface characteristics, as well as the electrostatic potential surface, were examined to understand the interaction between the (-)-Spirobrassinin and the PSBD1. Our findings indicate that (-)-Spirobrassinin predominantly binds to the hydrophobic grooves of PSBD1. Additionally, the binding of (-)-Spirobrassinin occurs at the interface between the positively and negatively charged regions of PSBD1, influenced by the charge properties of (-)-Spirobrassinin itself (Figure 5A,B). During the equilibrium simulations, we conducted a detailed analysis of the specific interactions between (-)-Spirobrassinin and PSBD1. Our analysis revealed the presence of hydrogen bonding, hydrophobic interactions, and van der Waals contacts between (-)-Spirobrassinin and PSBD1, as well as hydrogen bonding interactions with water molecules in the simulation system. Specifically, the benzene ring of (-)-Spirobrassinin formed a \(\pi\)-\(\pi\) stacking interaction with the phenylalanine residue Phe270, with a distance of 5.4 A. The carbonyl group of (-)-Spirobrassinin formed a hydrogen bond with the backbone amide of Phe261, with a distance of 2.1 A. The amino group on the ring of (-)-Spirobrassinin formed a hydrogen bond with a water molecule at a distance of 2.7 A, and the unsaturated nitrogen formed a hydrogen bond with another water molecule at a distance of 2.0 A. Furthermore, \begin{table}
\begin{tabular}{c c} \hline \hline
**Residue Name** & **RMSF(Å)** \\ \hline ALA & 14.468 \\ ASN & 14.033 \\ GLU & 12.360 \\ GLY & 12.329 \\ ARG & 11.412 \\ PRO & 11.190 \\ VAL & 10.923 \\ HIS & 10.758 \\ GLU & 10.574 \\ GLU & 10.338 \\ LEU & 10.268 \\ GLY & 10.096 \\ ASN & 9.928 \\ PRO & 9.707 \\ GLU & 9.427 \\ ASP & 8.934 \\ PHE & 8.914 \\ TRP & 8.766 \\ ARG & 8.653 \\ VAL & 8.521 \\ ILE & 8.452 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Flexible RMSF (Root Mean Square Fluctuation) of Amino Acid Residues. (-)-Spirobrassinin exhibited hydrophobic interactions with nearby hydrophobic amino acids, including Leu209, Ile213, Phe257, Ala260, Trp253, Met246, and others (Figure 5C,D). The Analysis of the (-)-Spirobrassinin-PSBD1 Contacts Revealed the Types and Quantities of Amino Acids Involved  
The analysis revealed that the main interactions between (-)-Spirobrassinin and PSBD1 were hydrogen bonding and \(\pi\)-\(\pi\) stacking. Hydrophobic interactions were not quantified but were determined based on the types of amino acids involved. The yellow color indicates the presence of contact, while the blue color indicates the number of interacting amino acid residues (Figure 6A). A consistent presence of yellow blocks throughout the 50 ns simulation for an amino acid suggests continuous interaction with (-)-Spirobrassinin. For example, Ile259 exhibited continuous blocks from 10 ns to 48 ns, indicating persistent hydrophobic interactions since this amino acid does not participate in hydrogen bonding. Similar analyses were performed for other amino acids. Notably, Phe261 and Phe270 showed a high frequency of continuous color blocks, indicating their critical roles in the interaction (Figure 6B). Figure 5: Analysis of the binding mode between the (-)-Spirobrassinin (green) and the PSBD1 during stable simulations. (**A**) Electrostatic potential surface of the PSBD1, with red indicating negatively charged regions and blue indicating positively charged regions; (**B**), distribution of hydrophobic and hydrophilic regions in the PSBD1, with green representing hydrophobic regions and magenta representing hydrophilic regions; (**C**), 3D interaction diagram, with hydrogen bonds shown as yellow dashed lines and the corresponding bond distances displayed. \(\pi\)-\(\pi\) stacking interactions represented by green dashed lines; and (**D**) 2D interaction diagram, with green representing hydrophobic amino acids, cyan representing polar amino acids, and gray representing the water molecules.