Upon initiation of apoptosis, the BCL-2 family proteins BAK and BAX oligomerise on the mitochondrial outer membrane, disrupting the membrane bilayer and forming pores. This process releases cytochrome c — a crucial juncture that commits the cell to death and despite decades of research is still poorly understood. We have designed binders the first potent and selective binders of BAK and BAX. Despite decades of research, this process, and the independent functions for BAK and BAX remain poorly understood, in part due to the lack of available tools that are selective for only BAK or BAX.
To overcome this limitation, in collaboration with the Baker lab (Institute for Protein Design) we used Rosetta in silico methods and directed evolution to design BAK and BAX binders. The proposed binding mode was validated by experimental crystal structures BAK binder complexes with BAK. These structures revealed unexpected conformational changes in BAK, which was distinct from the design model despite binding as predicted in the BAK binding groove. These conformational changes resembled the unfolding required for BAK to oligomerise on membranes. Further in vitro studies showed that at relatively low concentrations, the BAK binders induce BAK oligomerisation and membrane permeabilisation. In contrast, at higher concentrations, a threshold is crossed, inhibiting BAK oligomerisation. The same trend is observed for the BAX binders, with an altered threshold such that activation is dominant.
The BCL-2 family proteins are established drug targets, with first generation drugs transforming outcomes for blood cancer patients. Despite their significant target potential, there are currently no therapies that directly target BAK or BAX. Our findings have broad implications for designing effective BAK and BAX targeting small molecules and reveal potential uses for this generation of binders in disease therapies.