Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

alpha-Hemolysin (alpha-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, alpha-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to alpha-HL. Due to the binding of those compounds, the conformational transition of alpha-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of alpha-HL. However, these results have not indicated how the binding of the alpha-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of alpha-HL, could bind to the stem region of alpha-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of alpha-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the stem region of alpha-HL, the conformational transition of alpha-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of alpha-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.