Oral Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Antibodies targeting the α1-α2 loop of pro-apoptotic effectors Bak and Bax induce or inhibit apoptosis-associated conformation change and pore formation (#27)

Sweta Iyer 1 , Adeline Robin 1 , Abdullah Alazawi 1 , Richard Birkinshaw 1 , Melissa Shi 1 , Peter E Czabotar 1 , Peter Colman 1 , Ruth M Kluck 1
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia

Bak and Bax, pro-apoptotic members of the Bcl-2 family are required for mitochondrial pore formation during apoptotic cell death. Whereas Bak is a mitochondrial outer membrane protein, Bax is predominantly cytosolic in healthy cells. Both proteins become activated (i.e. conversion into a pore-forming protein) by the binding of BH3-only relatives at a hydrophobic surface groove (α2-α5). We recently identified an antibody (7D10) that can also directly activate Bak by binding to the α1-α2 loop. Binding was followed by displacement of the Bak α1 helix, as revealed by biochemical studies and a structural model of Fab bound to Bak. To examine the possibility of developing the antibody as therapeutic, smaller derivatives of 7D10 (Fab and scFv) were generated and found to trigger Bak activation in biochemical assays, and in cells. Ongoing studies are examining approaches for intracellular delivery of functional 7D10 fragments.

Intriguingly, antibodies to the Bax α1-α2 loop could also unfold α1 as revealed by crystal structures, but blocked Bax translocation to mitochondria. Conformation changes detected by limited proteolysis suggested that antibody binding on one side of the molecule (α1-α2 loop) allosterically altered the other side of the molecule to prevent exposure of the C-terminal transmembrane domain.

Thus, our study is the first to identify an antibody that induces global protein unfolding in Bak to induce apoptosis, as well as antibodies that inhibit Bax-mediated apoptosis. These antibodies present new tools to study Bak and Bax function, and avenues for the development of novel antibody formats to modulate apoptosis.