Dissecting the Structure of Oligomeric BAK and the Implications for Reactivating Apoptosis in Cancer

PROJECT SUMMARY Mitochondrial apoptosis is regulated by the pro- and anti-apoptotic members of the BCL-2 family, whose protein interactions determine whether a cell will live or die in response to distress. Cancer cells hijack the pathway, overexpressing anti-apoptotic members to suppress the pro-apoptotic members, thereby ensuring cellular immortality. Indeed, apoptotic suppression is a cardinal feature of oncogenesis and chemoresistance. BAK is one of two essential executioner proteins of the BCL-2 family and resides in a latent state in the mitochondrial outer membrane. When triggered by stress signals, BAK undergoes a conformational transformation, self-associates, and permeabilizes the mitochondrial outer membrane, resulting in the release of apoptogenic factors that commit the cell to apoptosis. How full-length BAK self-associates into an oligomer remains a mechanistic mystery and is the focus of my F32 application. The Walensky laboratory reported the generation of the first stable and homogeneous oligomeric species of full-length BAX, the cytosolic homolog of BAK, and characterized it through a battery of structure-function analyses. In contrast to BAX, which undergoes a cytosol to mitochondrial translocation upon triggering, BAK is constitutively localized in the mitochondria as a membrane-embedded protein. As such, BAK has been challenging to express in full-length form, and with sufficient purity and yield to embark on parallel studies. Encouraged by the success of generating and characterizing a full-length BAX oligomer, I applied key learnings to produce a stable, full-length oligomeric species of BAK (BAKO) for the first time, setting the stage for my postdoctoral studies on dissecting the structure and function of BAKO. My goal is to generate fresh insight into the structure-function mechanism of BAK-mediated mitochondrial apoptosis and leverage the molecular details of the oligomeric interfaces to inform new opportunities for enhancing BAK oligomerization and apoptosis induction in treatment-resistant cancer. Thus, I aim to (1) develop and characterize nanobodies that bind to oligomeric BAK and (2) harness BAKO-binding nanobodies to determine the structure of oligomeric BAK and the binding interfaces critical to its membrane- permeabilizing function. To accomplish my research aims, I will undertake a multidisciplinary workflow that incorporates a nanobody discovery platform, protein engineering, biochemical assays in model membranes and mitochondria, a series of structural methods, and mechanistic analyses of apoptosis in cancer cells. In pursuing this research plan, which includes a series of alternative approaches, I aim to both characterize the execution-phase of BAK-mediated apoptosis and uncover novel and potentially druggable surfaces for therapeutic benefit in cancer. I am excited to be pursuing a comprehensive postdoctoral training program in the laboratories of Dr. Loren Walensky at the Dana-Farber Cancer Institute and Dr. Andrew Kruse at Harvard Medical School, and look forward to developing as an independent and innovative scientist at the intersection of biochemistry, structural biology, and cancer biology.