Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structure of the BAK-inhibiting antibody 14G6 bound to BAK identifies core-latch separation and α1-dissociation as essential events in BAK activation (#406)

Michelle S Miller 1 , Hema Preethi Subas Satish 1 , Sweta Iyer 1 , Ahmad Wardak 1 , Daisy Lio 1 , Melissa X Shi 1 , Agnes Wong 1 , Rachel Uren 1 , Jason Brouwer 1 , Peter Czabotar 1 , Ruth Kluck 1
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia

Apoptosis, a form of programmed cell death regulated by the BCL-2 family of proteins, plays a vital role in early development by removing unwanted cells as well as an ongoing role in preventing cancer. The drug venetoclax, which targets the pro-survival family member, BCL-2, induces apoptosis and has shown great promise in treating cancer. On the other end of the spectrum, inhibitors of apoptosis have the potential to prevent unwanted cell death in a range of conditions including hearing loss, ischemic reperfusion injury and traumatic brain injury. Here we report the first antibody inhibitor of the pro-apoptotic family member BAK. The 14G6 rat monoclonal antibody inhibits BAK activation and oligomerisation triggered by three distinct BAK activators: cBID, the 7D10 antibody and heat. The crystal structure of 14G6 Fab bound to human BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites previously reported to be involved in BAK activation. Biochemical and biophysical experiments support a two-pronged mechanism where binding of both the α1 helix and α5-α6 hinge contribute to the inhibition of both activation and oligomerisation of BAK. This supports crucial roles for dissociation of α1 and for separation of the core (α2- α5) and latch (α6- α9) regions in the process of BAK activation. 14G6 is the first antibody specific for non-activated BAK, and the first inhibitor of human BAK.