Anti-cancer drugs and cellular stresses induce mitochondrial apoptosis by upregulating or activating pro-apoptotic BH3-only members of the Bcl-2 protein family. The eight BH3-only proteins have different abilities to activate the pore-forming proteins Bak and Bax, and different binding affinities for pro-survival Bcl-2 family members. However, the study of their binding profiles has been largely limited to the use of peptides based on the BH3 ‘ligand’ domain because all BH3-only proteins, except Bid, are largely unstructured and unstable as recombinant proteins.
To clarify the ability of BH3-only proteins to activate Bak and Bax on mitochondria we developed stable recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). While peptides may have differences in biophysical properties such as helicity and hydrophobicity that are difficult to control, Bid chimeras provide a robust scaffold to present the BH3 domain to binding partners. These Bid chimeras were around 1000-fold more effective at activating Bak and Bax than the equivalent BH3 peptides. Thus, Bid BH3 chimeras are important new tools to examine Bcl-2 family function. The results indicate that Bid and Bim are the strongest activators but that all other BH3-only proteins except for Bad and Noxa are also activators to a lesser extent.