Structural investigations of pro‑apoptotic Bcl‑2 family proteins

© 2017 Dr. Angus David CowanThe Bcl‑2 protein family regulates the intrinsic apoptotic pathway through an intricate network of protein:protein and protein:membrane interactions. The pathway culminates in the permeabilisation of the mitochondrial outer membrane by the pro‑apoptotic effector proteins Bak and Bax, an event that irreversibly commits a cell to death. To facilitate membrane permeabilisation, Bak and Bax undergo a series of conformational changes to convert from inert monomers to membrane‑embedded homodimers that nucleate and propagate apoptotic oligomers. While great strides have been made in structurally characterising these conformational changes, questions remain surrounding homodimer interactions with the membrane, oligomerisation, and membrane pore formation. This thesis addresses these questions by providing structures of lipids bound to Bak BH3:groove core homodimers (Chapter 2). These are the first structures of any Bcl‑2 family protein in complex with lipid. They reveal symmetric binding sites for phospholipid headgroups and acyl chains. In one structure, adjacent Bak homodimers are cross‑linked by the acyl chains of single phospholipids, suggesting homodimer oligomerisation could be mediated by lipid. Bak oligomers could be dissociated with phospholipase A2, supporting a role for lipid in oligomer stability. Collectively, the structures presented here indicate that lipids may play a direct role in Bak oligomerisation. Like Bak, Bax homodimerises and oligomerises on the mitochondrial outer membrane. The original Bax BH3:groove core homodimer structure was solved as a GFP fusion at low resolution. Here, a tetrameric structure consisting of two Bax BH3:groove core homodimers alone was solved at high resolution (Chapter 3), providing details for canonical interactions in atomic detail. A crystal structure of Bax BH3:groove core homodimers containing lipid was also solved, although the structure could not be refined due to severe twinning. This result demonstrates that Bax core domains also associate with lipid, and provides a starting point for crystal optimisation. Pro‑survival Bcl‑2 family proteins antagonise the apoptotic function of Bak and Bax by preventing their activation and sequestering their activated forms. Sequestration of activated Bak and Bax in heterodimeric Mode 2 complexes involves binding of the Bak/Bax BH3 domain to a conserved hydrophobic groove. Beyond this, little is known regarding the topology of these complexes. The pro‑survival protein Bcl‑XL can undergo similar conformational changes to Bak and Bax, but whether it forms BH3:groove heterodimers with Bak/Bax was unknown. Using cysteine cross‑linking on mitochondria, I show that Bcl‑XL can form reciprocal BH3:groove heterodimers with Bax, and possibly Bak (Chapter 4). These results challenge a simplistic view of Mode 2 complexes, implicating more extensive interactions beyond the canonical BH3 in groove interface. Bok is a third potential pro‑apoptotic effector protein that shares sequence similarity with Bak and Bax, but its role in apoptosis remains unresolved. To investigate the structure and function of Bok, I developed a recombinant expression system to produce human, rat, and chicken Bok. The first crystal structure of Bok, from the chicken, reveals the canonical Bcl‑2 family fold, with deviations that may explain its proposed constitutive activity (Chapter 5). The structure paves the way for mutagenesis studies that will further our understanding of this enigmatic protein