Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use a similar binding mode and are not distinguished by binding affinity or kinetics

Abstract

Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, proapoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space.