Mode of action and structural modelling of the interaction of formononetin with suilysin

Suilysin is a critical pore-forming virulence factor of Streptococcus suis that has been demonstrated to substantially contribute to its pathogenicity. We have demonstrated that formononetin alleviates S. suis infection both in vivo and in vitro by targeting suilysin. However, the molecular mechanism of the effect is unclear. Our aim was to determine the molecular mechanism of the effect of formononetin on suilysin.

The mechanism of interaction between formononetin and suilysin was investigated by molecular modelling. The results indicated that formononetin was bound at the junction of domain two and domain four of suilysin. The binding free energy values indicated that the A415, Y412, E414, N413, T61, T62 and G416 residues are critical for this binding, this observation was confirmed by the changes in the flexibility of these residues and the distances between these residues and formononetin. The inhibitory effect of formononetin on the pore-forming activity of suilysin, binding constant and binding free energy were significantly decreased by site-specific mutagenesis of Y412 and N413. Finally, we analysed the spatial configuration of suilysin before and after formononetin binding, the results indicated that the binding changed the conformation of suilysin, especially the angle between domain two and domain four, resulting in the disruption of cholesterol binding to suilysin and in the loss of pore-forming activity.

Formononetin is located at the junction of domain two and domain four of suilysin, and Y412 and N413 play critical roles in the binding. Formononetin binding changes the angle between domain two and domain four of suilysin, resulting in the loss of the pore-inducing activity of suilysin.

This work will promote the application of formononetin to combat S. suis infections and may contribute to the development of new inhibitors or modification of existing inhibitors.