Abstract
BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.
Highlights
BCL-2 proteins regulate mitochondrial poration in apoptosis initiation
At 200 and 400 nM BAK spontaneously permeabilizes liposomes, suggesting its autoactivation; at lower BAK levels direct activation by BID BCL-2 homology 3 (BH3) is required for permeabilization (Fig. 1a, b and Supplementary Fig. 1a, b)
Autoactivation cooperates with direct activation to amplify signaling and lower the threshold of BAK required for mitochondrial poration (Supplementary Movie 2)
Summary
BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity. Mitochondrial poration by pore-forming BCL-2 Effectors, BAK and BAX, initiates apoptosis[1,2,3]. BIM BH3 peptide ligand complexes with BAK (and similar complexes with BAX17,33) have revealed cavities that form at the bottom of the activation groove at the interface with helix α1, which reflect destabilization of Effectors assumed to promote BAK and BAX activation[27]. Designed inhibitory BIM BH3 peptide ligands “glued” through nonnatural amino acids-mediated salt bridges to helix α1 at the bottom of the activation groove block BAK activation and membrane poration, implicating this region in BAK activation or inactivation[27]. The core dimers may oligomerize through latch-mediated dimerization to porate the outer mitochondrial membrane[3,7,28,30,37,38] as well as through bridging by phospholipids[39], the mechanism of poration has not been elucidated
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