title: Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.
abstract: The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.
authors.affiliation: From the Departments of Biochemistry and Medicine, Albert Einstein Cancer Center, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Onyinyechukwu
authors.initials: O
authors.lastname: Uchime
authors.affiliation: the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Dai
authors.affiliation: From the Departments of Biochemistry and Medicine, Albert Einstein Cancer Center, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Biris
authors.affiliation: the School of Medicine, University of California, San Diego, San Diego, California 92093, and.
authors.forename: David
authors.initials: D
authors.lastname: Lee
authors.affiliation: the Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: the School of Medicine, University of California, San Diego, San Diego, California 92093, and.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.affiliation: the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, jon.lai@einstein.yu.edu.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Lai
authors.affiliation: From the Departments of Biochemistry and Medicine, Albert Einstein Cancer Center, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, New York 10461, evripidis.gavathiotis@einstein.yu.edu.
authors.forename: Evripidis
authors.initials: E
authors.lastname: Gavathiotis
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D001665:Binding Sites; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000906:Antibodies; D051023:BH3 Interacting Domain Death Agonist Protein; D007140:Immunoglobulin Fab Fragments; D008081:Liposomes; D050505:Mutant Proteins; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c
keywords: B cell lymphoma 2 (Bcl-2) family; BAX; antibody engineering; mitochondrial apoptosis; phage display; protein structure; protein targeting; structure-function; synthetic antibody