Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization

Abstract

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak-BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak-BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein-protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.

Figure 1.

Direct interaction of Bim_EL, tBid, and Noxa with Bak. (A and D) SPR (relative units [RU]) of immobilized full-length wt human Bim_EL (A) or Noxa (D) exposed to increasing concentrations of purified BakΔTM. (B) K_ds of various BH3-only proteins and BakΔTM calculated from SPR assays. Error bars are ± SD of three independent experiments using different chips and protein preparations. (C) SPR of immobilized BakΔTM exposed to 200 nM purified NoxaΔCT, BadΔCT, or tBid. (E) Purified S peptide–Noxa was mixed with His₆-Bak or GST-Bak and then recovered on S protein–agarose as described in the Materials and methods. The pull-downs and one-fifth of the inputs were subjected to SDS-PAGE and immunoblotting. Molecular masses are given in kilodaltons. (F and G) SPR of immobilized full-length wt Bim_EL (F) or Noxa (G) exposed to purified BakΔTM in buffer ± 1% CHAPS. (H) K_ds in the absence or presence of CHAPS. Error bars are ± SD of three experiments using different protein preparations. (I) Effect of mitochondrial lipids on Noxa–Bak binding. WB, Western blot.

Figure 2.

BH3 domain–Bak interactions involve the Bak BH3-binding groove. (A) Sequence alignment of selected human BH3 domains. Residues marked in red are mutated in NoxaΔCT 3E and NoxaΔCT 2A. (B) SPR (relative units [RU]) of immobilized BakΔTM exposed to 200 nM purified NoxaΔCT, NoxaΔCT 3E, or NoxaΔCT 2A. (C) Sequence alignment of selected human BH1 domains. The asterisk indicates the conserved BH3-binding groove Arg that is critical for the function of antiapoptotic family members (Sattler et al., 1997). (D) SPR (relative units) of immobilized full-length Bim_EL exposed to 200 nM BakΔTM or BakΔTM R127A in CHAPS. (E) Alignment of Bax and Bak showing Bax Lys21 (asterisk) implicated in BH3 peptide binding. (F) SPR of immobilized Noxa exposed to 400 nM BakΔTM or BakΔTM R36A. (G) Top view of Bak structure (Moldoveanu et al., 2006) used as a starting point for docking the Noxa BH3 domain and performing MDDSs. Note the blockage of the BH3-binding groove by Y89^Bak and R88^Bak (red). (H) Top view of Noxa (in helix model) in complex with Bak (in surface model). (I) Top view of Bak in the bound state showing conformational changes of Y89^Bak and R88^Bak to vacate the Noxa-binding groove with two hydrophobic holes. (J) SPR of immobilized Noxa exposed to 800-nM fusions of GST with Bak, Bak 127A, Bak G126S, Bak 93E, Bak 114E, Bak 93E/114E, or GST alone. (K) SPR of immobilized full-length Bim_EL exposed to 200 nM BakΔTM or BakΔTM G126S. In panels A, C, and E, identical residues are indicated by black shading, and similar residues are shown by gray shading.

Figure 3.

Bak oligomerization depends on BH3 domain–BH3-binding groove interactions. Reactions containing purified Bak (A–H), Bak R127A (I), or Bak R36A (J) alone or with a threefold molar excess of Bad (A), Bim_EL (B), tBid (C), Noxa (D, E, G, I, and J), or Noxa 2A (H) or a 1:10 molar ratio of Noxa to Bak (F) was incubated for 20 min (D), 24 h (F), or 1 h (other panels) at 23°C in the absence (G) or presence of DTT (all other panels) and then subjected to pull-down and blotting (G, right) or applied to a Superdex S200 column (other panels). Column fractions were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted (A, B, and F) or silver stained (other panels). Arrows indicate size markers. Red boxes show oligomerized Bak. Green boxes show monomeric BH3-only proteins in reactions in which Bak oligomerized. The orange box shows transient intermediate complexes.

Figure 4.

Bak membrane permeabilization depends on BH3 domain–BH3-binding groove interactions. (A) Purified Bak and Noxa at the indicated molar ratios were incubated for 1 h in the presence of CHAPS and captured using antiactive Bak Ab-1 precoupled to protein A/G–agarose. Immunoprecipitates and half of the inputs were blotted with an antibody against total Bak. (B) Peptides used to trigger Bak activation in vitro. Residues mutated in various peptides are indicated in red. (C–E) FITC–dextran 10 encapsulated in LUVs containing 5% DGS-NTA-Ni was incubated with 500 nM (C and D) or 1 µM (E) His₆-BakΔCT along with 1 µM Bim BH3 peptide, 1 or 20 µM Bad BH3 peptide, or 2 µM of the indicated Noxa BH3 peptide. FITC-dextran release was measured as a function of time (C) or calculated at 4,000 s from three independent experiments (D and E). Error bars are means ± SD. (F) Liposomes were incubated with 500 nM Bak and the indicated BH3 peptide (1 µM) at 37°C for 30 min, then cross-linked with 100 µM BMH, and analyzed. (G and H) Liposomes were incubated with 2 µM His₆-BakΔCT G126S or His₆-BakΔCT N86G/G126S in the absence or presence of a 10-fold molar excess of the indicated Bim BH3 peptide. FITC-dextran release was measured as a function of time (G) or calculated at 4,000 s from two independent experiments (H). (I) After mitochondria from Bax^−/−Bak^−/− MEFs were incubated with purified proteins for 1 h, sedimented, and washed, the supernatant and pellet were blotted for cytochrome c (Cyto C) and mitochondrial Hsp60. Molecular masses are given in kilodaltons. IP, immunoprecipitation; IB, immunoblot.

Figure 5.

Bim- and Noxa-induced cytotoxicity depends on BH3 domain–BH3-binding groove interactions. (A) Whole-cell lysates prepared from wt MEFs, Bax^−/−Bak^−/− MEFs or Bax^−/−Bak^−/− MEFs reconstituted with the indicated Bak constructs were harvested for immunoblotting. #1 and #2 represent independent clones. Molecular masses are given in kilodaltons. (B) MEFs were transfected with empty vector or the indicated BH3-only protein and assayed for colony formation. The number of colonies obtained from cells transfected with control plasmid (pSPN) was set to 100%. Error bars are means ± SD of three independent experiments. DKO, double knockout.