Analysis of the Binding Sites on BAX and the Mechanism of BAX Activators through Extensive Molecular Dynamics Simulations.

The BAX protein is a pro-apoptotic member of the Bcl-2 family, which triggers apoptosis by causing permeabilization of the mitochondrial outer membrane. However, the activation mechanism of BAX is far from being understood. Although a few small-molecule BAX activators have been reported in the literature, their crystal structures in complex with BAX have not been resolved. So far, their binding modes were modeled at most by simple molecular docking efforts. Lack of an in-depth understanding of the activation mechanism of BAX hinders the development of more effective BAX activators. In this work, we employed cosolvent molecular dynamics simulation to detect the potential binding sites on the surface of BAX and performed a long-time molecular dynamics simulation (50 μs in total) to derive the possible binding modes of three BAX activators (i.e., BAM7, BTC-8, and BTSA1) reported in the literature. Our results indicate that the trigger, S184, and vMIA sites are the three major binding sites on the full-length BAX structure. Moreover, the canonical hydrophobic groove is clearly detected on the α9-truncated BAX structure, which is consistent with the outcomes of relevant experimental studies. Interestingly, it is observed that solvent probes bind to the trigger bottom pocket more stably than the PPI trigger site. Each activator was subjected to unbiased molecular dynamics simulations started at the three major binding sites in five parallel jobs. Our MD results indicate that all three activators tend to stay at the trigger site with favorable MM-GB/SA binding energies. BAM7 and BTSA1 can enter the trigger bottom pocket and thereby enhance the movement of the α1-α2 loop, which may be a key factor at the early stage of BAX activation. Our molecular modeling results may provide useful guidance for designing smart biological experiments to further explore BAX activation and directing structure-based efforts toward discovering more effective BAX activators.