Domain-specific insight into the recognition of BH3-death motifs by the pro-survival Bcl-2 protein

Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryo -genesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lym-phoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, can-cers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molec-ular basis of Bcl-2's ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane -mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2's functional domains engage in recognizing and inhibiting apoptotic proteins via their prom-inent BH3 motifs. This way, we provide a unique residue-and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

Automated summary

Programmed mammalian cell death is regulated by the Bcl-2 protein, which plays a crucial role in recognizing and inhibiting apoptotic proteins to protect cells. By using nuclear magnetic resonance, the molecular functioning of Bcl-2 at the membrane level was studied, providing insights into its residue-specific response and affinity.