First structural Elucidation of Bcl-2 functional conversion induced by validated modulators using microsecond-scale molecular dynamics simulations and density functional theory.

Cancer remains a leading cause of mortality worldwide, largely driven by uncontrolled cell proliferation and the evasion of apoptosis, a tightly regulated process essential for cellular homeostasis. The Bcl-2 family of proteins plays a central role in regulating the intrinsic apoptotic pathway, with anti-apoptotic Bcl-2 promoting cell survival by sequestering pro-apoptotic Bax. Although Bcl-2 functional converters (BFCs) have been experimentally validated to reprogram Bcl-2 toward a pro-apoptotic function, the molecular-level structural and dynamic mechanisms underlying this functional conversion remain poorly understood. In particular, direct atomistic evidence explaining how targeting the intrinsically flexible loop domain drives conformational switching in Bcl-2 is still lacking. To address this critical gap, the present study provides the first structural and dynamic insights into FLD-mediated functional conversion of Bcl-2 using representative synthetic, experimentally validated BFCs (BFC1103 & BFC1108). Molecular docking followed by atomistic microsecond-scale MD simulations was employed to delineate binding stability and conformational changes. Extensive docking analyses revealed strong binding affinity and persistent non-covalent interactions with key FLD residues. Long-timescale atomistic MD simulations demonstrated that FLD engagement induces pronounced conformational changes in Bcl-2, resulting in BH3 domain exposure and a Bax-like, pro-apoptotic structural state. Collectively, this work establishes a first-of-its-kind structural frame work that links FLD targeting to Bcl-2 functional conversion, thereby bridging experimental observations with atomistic simulations and providing a rational basis for the design of next-generation apoptosis-restoring anticancer therapeutics.