Nanobodies for Dissecting the Structure and Function of Oligomeric BAX

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PROJECT SUMMARY BCL-2 family proteins are critical regulators of apoptosis and deregulation of their protein interaction network drives oncogenesis and chemoresistance. BAX is a pro-apoptotic BCL-2 protein that serves as a cardinal executioner of the death pathway. During homeostasis, BAX resides as a latent monomer in the cytosol until triggered by cellular stress to undergo a major conformational change, leading to its translocation to the mitochondria and self-assembly into oligomeric species that permeabilize the mitochondrial outer membrane. Cancer cells usurp the survival arm of the pathway, overexpressing anti-apoptotic members such as BCL-2 and MCL-1, which can trap activated monomers of BAX, prevent oligomeric assembly, and thereby preserve mitochondrial integrity. A critical missing link in our understanding of BAX-mediated apoptosis during homeostasis and cancer is the structure of oligomeric BAX, referred to as the “holy grail” of apoptosis research. The Walensky laboratory has recently generated the first stable and homogeneous oligomeric species of full- length BAX amenable to structure-function analyses. Having characterized this BAX oligomer, termed BAXo, by small-angle X-ray scattering, negative stain electron microscopy (EM) of BAX-porated liposomes, and comparative functional studies of wild-type and BAX mutants in liposomes, mitochondria, and cells, a critical next step is to deploy BAXo to solve a definitive structure and interrogate its functional interfaces. I hypothesize that by generating diverse nanobodies against BAXo, I will be able to obtain high-resolution structures of this elusive “death channel” and generate fresh insight into the mechanism of BAX-mediated mitochondrial apoptosis. Specifically, I aim to (1) develop and characterize nanobodies that bind to oligomeric BAX and (2) harness BAXo- binding nanobodies to determine the structure of oligomeric BAX and the interfaces critical to membrane- permeabilizing function. To accomplish my goals, I will pursue a multidisciplinary workflow that incorporates a yeast display nanobody discovery platform, protein engineering, biochemical assays in model membranes and mitochondria, hydrogen-deuterium exchange mass spectrometry, X-ray crystallography, cryo- EM microscopy, and mechanistic analyses of apoptosis in cancer cells. Thus, by developing and deploying BAXo-binding nanobodies in comprehensive structure-function studies with built-in alternative approaches, I aim to both characterize the execution-phase of BAX-mediated apoptosis and uncover novel and potentially druggable surfaces for therapeutic benefit in cancer. I am excited to be pursuing a rigorous graduate training program in the laboratory of Dr. Loren Walensky at the Dana-Farber Cancer Institute and Harvard Medical School, and look forward to developing as an independent and innovative physician-scientist at the interface of biochemistry, structural biology, cancer biology, and clinical oncology.