The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

The BCL-2 family proteins are key positive and negative regulators of apoptosis. Biochemical and subcellular fractionation studies localize the anti-apoptotic members, such as BCL-2 and BCL-X L , at outer mitochondrial membranes (OMM) 1 and, to a lesser extent, at endoplasmic reticulum and nuclear membranes. Pro-apoptotic factors either reside at the OMM or as soluble cytosolic proteins, as with BAX, BID, and BAD. Following an apoptotic signal, these factors are translocated to mitochondria. BCL-2 and BCL-X L cancel out the activity of pro-apoptotic members at the OMM, at least partly through direct interactions (1).The tertiary structures of pro-and anti-apoptotic BCL-2 proteins consist of all ␣-helical protein folds involving one or more conserved BCL-2-homology domains (BH1-4). Although all four BH domains are necessary for the proper functioning of BCL-2 and BCL-X L , the BH3 domains of pro-apoptotic members are themselves sufficient for apoptosis induction (2). BH3 domains from pro-apoptotic BH proteins also make heterodimer contacts with related anti-apoptotic partners. The structure of the complex of a soluble BCL-X L protein (BCL-X L ⌬C) and BAK BH3 peptide from nuclear magnetic resonance (NMR) spectroscopy showed peptide bound to a hydrophobic groove bounded by the BH1, BH2, and BH3 domains of BCL-X L (3).In addition to their ability to nullify pro-apoptotic binding partners, the anti-apoptotic BCL-2 family members may maintain cell survival through regulation of ion and metabolite gradients across mitochondrial membranes. BCL-X L and BCL-2 form pores in synthetic lipid vesicles and membranes. Interestingly, this intrinsic pore-forming activity is inhibited by a bound BAK BH3 peptide (4). It remains an unresolved question whether BCL-X L and BCL-2 affect OMM permeability directly or through interactions with other known mitochondrial channels. For example, BCL-X L may promote osmotic homeostasis by maintaining the voltage-dependent anion channel in an open state, allowing transit of metabolites across the OMM (5-7). BCL-X L also interacts with the adenine nucleotide translocator, the most abundant protein in the inner mitochondrial membrane and a major component of the permeability transition pore complex (8).Previously, we reported that AA, an inhibitor of mitochondrial electron transport at complex III, selectively kills murine hepatocyte cell lines that over-express BCL-X L or BCL-2 (4, 9). Mitochondrial electron transport inhibition is not correlated with differential killing of BCL-X L -expressing cells, as 2-methoxy AA (2-OMeAA) has similar effects despite a 1000-fold reduction in electron transport inhibition. Rather, a direct effect of AA on BCL-X L function(s) was proposed, based on fluorescent binding assays showing stoichiometric interaction of AA and BCL-X L (⌬C). Computational modeling using the BCL-X L (⌬C) NMR structure (3) predicted a binding site for AA along the long axis of the hydrophobic groove (Fig. 1). Finally, dosedependent inhibition of BCL-X L (⌬C) pores in synthetic liposome...

Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

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