Exploring the Activation Landscape of Pro-Apoptotic BAK Through the Discovery of Human BH3-Only and Non-Native Peptide Binders

Abstract

BAK is one of the two pro-apoptotic members that form part of the BCL-2 protein family. Previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, and PUMA to pro-apoptotic BAK leads to mitochondrial outer membrane permeabilization (MOMP), release of cytochrome c, and ultimately cell death. The BH3 binding event leads to a series of conformational changes that promote the conversion of BAK from monomer to dimer and subsequently to oligomers that disrupt membranes in a process referred to as activation. Putative intermediate crystal structures, 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 thesis, I address the question using three methods: 1) computational design, 2) yeast surface-display screening of candidate BH3-like peptides, and 3) structure-based energy scoring. I identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria in HeLa cells. These new activators expand current views of how BAK-mediated cell death can be triggered. I show binding and kinetics measurements and solved crystal structures of BAK-peptide complexes, including complexes for two inhibitors and one activator. Results reveal a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. Here, I propose a free energy model for BAK activation that is based on the differential engagement of BAK monomers and the BAK activation transition state that integrates observations described in this thesis and previous reports of BAK binders, activators, and inhibitors.