Combined Nudged Elastic Band and Markov State Models Uncover Distinct BAX Activation Pathways by Bim Peptide and a Small Molecule.

BAX, a key pro-apoptotic member of the BCL-2 family, represents a promising therapeutic target for overcoming apoptotic resistance in cancer. Although structural and biochemical studies have advanced our understanding, the detailed mechanism of BAX activation remains unresolved. Activation involves dynamic conformational changes, including ligand migration from the trigger site and release of the α9 helix, processes that are challenging to capture with conventional approaches. Here, we combine the NEB method with long-time scale MD simulations to effectively sample intermediate conformations along the BAX activation pathway induced by the Bim peptide or the small molecule 27c. Integrating MSMs with MFEP analysis, we uncover distinct activation pathways for Bim and 27c from both kinetic and thermodynamics perspectives. Our results provide atomic-level insights into the conformational transition of BAX from its inactive to active state, highlighting fundamental differences between peptide- and small molecule-mediated activation. These findings not only deepen the mechanistic understanding of BAX regulation but also offer a foundation for the rational design of next-generation small molecule activators.