Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the alpha 2-alpha 5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the alpha 1-alpha 2 loop. A monoclonal antibody (clone 7D10) binds close to alpha 1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to alpha 1. An antibody (clone 3C10) to the Bax alpha 1-alpha 2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates alpha 1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under alpha 1. Our identification of the alpha 1-alpha 2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.