The functional differences between pro-survival and pro-apoptotic B cell lymphoma 2 (Bcl-2) proteins depend on structural differences in their Bcl-2 homology 3 (BH3) domains

Abstract

Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity "BH3-in-groove" complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.

Keywords: Apoptosis; B cell Lymphoma 2 (Bcl-2) Family; Cell Death; Peptides; Protein Structure.

FIGURE 1.

Pro-survival BH3 domains bind pro-survival proteins poorly. A, alignment of BH3 domains from pro-apoptotic and pro-survival Bcl-2 family members. The four conserved hydrophobic residues h1–h4 are indicated (blue). Residues that distinguish pro-survival versus pro-apoptotic BH3 domains, h1 + 1 and h3, are shaded gray. Shown is killing activity of Bim_S chimeras with the Bcl-x_L (B), Bcl-2 (C), or Bcl-w BH3 (D) domains and their h1 + 1/h3 (mt3) mutants in MEFs. E, Western blot showing relative expression of FLAG-tagged Bim_S and Bim_S chimeras expressed in Bax^−/−/Bak^−/− MEFs. F, killing activity of the Bcl-2 BH3 domain, Bim_S (positive control), or Bim_S4E (negative control) in MEFs co-expressing Noxa (to neutralize Mcl-1) or Bim_S4E as controls. Colonies were scored, and numbers were expressed as a percentage of the number observed in cells transduced with Bim_S4E alone. G, co-immunoprecipitation of endogenous Mcl-1 from Bax^−/−/Bak^−/− MEFs expressing FLAG-tagged Bim_S pro-survival BH3 chimeras. Western blots of immunoprecipitates (IP) and whole cell lysates (WCL) were probed with anti-Mcl-1 and anti-FLAG antibodies. H, Bim_S chimeras with mutant Bak BH3 domains fail to kill wild-type MEFs, unlike wild-type Bim_S or a chimera with the wild-type Bak BH3 sequence. Error bars, S.D. of n = 2–3 separate assays. In B–D, F, and H, colonies were scored 7 days after transduction, and numbers are expressed as a percentage of the number observed in cells transduced with an inert Bim_S mutant (Bim_S4E).

FIGURE 2.

Cytochrome c release assay with Bcl-x_L BH3 peptides. Permeabilized MEFs were treated with the indicated concentrations of synthetic peptides corresponding to the wild-type Bcl-x_L BH3 domain (wt), the wild-type Bcl-x_L BH3 domain fused to a mitochondrial targeting sequence (wt-MTS), or the Bcl-x_L h1 + 1/h3 mutant BH3 domain fused to a mitochondrial targeting sequence (mt3-MTS) (left) or the wild-type Bim BH3 domain (wt) or the wild-type Bim BH3 domain fused to a mitochondrial targeting sequence (wt-MTS) (right). The presence of cytochrome c released into the soluble fraction following peptide treatment was determined by Western blotting (WB) with an anti-cytochrome c antibody.

FIGURE 3.

Killing activity of Bcl-2ΔN34 in MEFs. Bcl-2ΔN34 only kills MEFs in the absence of Mcl-1. Colonies were scored 7 days after transduction, and numbers were expressed as a percentage of the number observed in cells transduced with the vector alone. Error bars, S.D. of n = 2–3 separate assays.

FIGURE 4.

Crystal structure of Bcl-x_L bound to its own BH3 domain. A, overlay of the crystal structures of Bcl-x_L bound to its own BH3 domain (Bcl-x_L molecule A (mauve) and Bcl-x_L BH3 (green)) and bound to Bim BH3 (Bcl-x_L (light blue) and Bim BH3 (orange)). B, region of the electron density map showing the lysine at h1 + 1 in the Bcl-x_L BH3 peptide ligand. C, the N-terminal end of the Bcl-x_L BH3 peptide ligand (green) is displaced out of the binding groove relative to the Bim BH3 domain (orange). D, the h1 hydrophobic residue in the Bcl-x_L BH3 domain (green) is not buried in the binding groove, unlike the corresponding residue in Bim (orange). Similarly, hydrophobic contacts between Bcl-x_L and the h3 residue of the Bcl-x_L BH3 ligand are greatly reduced due to the small alanine residue here compared with the larger hydrophobic residue found in all pro-apoptotic BH3 domains (such as the isoleucine in Bim).

FIGURE 5.

BH3 domain mutations destabilize pro-survival proteins. A, pro-survival proteins with “pro-apoptotic” BH3 domains do not kill Bax^−/−/Bak^−/− MEFs, even when co-expressed with Bim_S. Colonies were scored, and numbers were expressed as a percentage of the number observed in cells transduced with the pMIG vector only. Error bars, S.D. of n = 3 assays. B, mutation of the h1 + 1, h3 or both residues in pro-survival proteins significantly impacts steady-state levels of FLAG-tagged pro-survival proteins in Bax^−/−/Bak^−/−MEFs. C, the reduced levels of pro-survival BH3 mutants are due to their shorter half-life as a result of them being more rapidly degraded by the proteasome following cycloheximide (CHX) treatment. In B and C, Western blots (WB) of equivalent cell lysates were probed with anti-FLAG antibody and then reprobed with anti-β-actin antibody to control for sample loading. The asterisk in C indicates a nonspecific band that becomes apparent due to the longer exposure of this blot.

FIGURE 6.

Structures showing interactions between the h1 + 1 residue of the BH3 domain on pro-survival proteins and the α1 helix. In Bcl-x_L (PDB code 1PQ0) (A), Bcl-2 (PDB code 2XA0) (B), and Bcl-w (PDB code 1O0L) (C), the h1 + 1 residue makes hydrogen bond/salt bridges with residues on the α1 helix, whereas in Mcl-1 (PDB code 1WSX) (D), the h1 + 1 leucine makes hydrophobic contacts.

FIGURE 7.

Interactions between α1 and α2 helices stabilize Bcl-x_L. A, close-up view of the Bcl-x_L crystal structure (PDB code 1PQ0) highlighting the network of interactions between residues on the α1 and α2 helices. B, mutation of residues on α1 or α2 helices impacts steady-state levels of Bcl-x_L in MEFs. A long (dark) exposure is provided so that expression of the E7A/D11A mutant can be seen. C, the reduced levels of the mutants are due to their shorter half-life as they are more rapidly degraded by the proteasome following cycloheximide (CHX) treatment. In B and C, Western blots (WB) of equivalent cell lysates were probed with anti-FLAG antibody and then reprobed with anti-β-actin antibody to control for sample loading. The asterisk in C indicates a nonspecific band that becomes apparent due to the longer exposure of this blot. D, Western blot of lysates of cells expressing full-length FLAG-tagged Bcl-x_L and FLAG-tagged Bcl-x_LΔN61. The asterisk indicates a nonspecific band.

FIGURE 8.

Crystal structure of Bcl-w with a “pro-apoptotic” BH3 domain. A, crystal structure of the Bcl-w dimer formed by reciprocal salt bridges between Arg-47 and Asp-51 on the excised Bcl-w BH3 domains (teal). The position of the excised Bcl-w BH3 domain (residues 38–58) within the intact Bcl-w protein is shown in blue. B, view of the excised BH3 domain (teal) within the canonical binding groove formed by helices α3-α4. C, Coomassie-stained SDS-polyacrylamide gel showing that Bcl-w with a mutant BH3 domain is extensively degraded following expression and purification from E. coli, unlike the wild-type protein. D, the overall structure of the Bcl-w BH3 mutant (mauve) is similar to native Bcl-w (yellow; PDB entry 1O0L without the C-terminal “tail” region removed) (37), although the α3 helix is highly disordered and the α2 helix is extended by one extra turn.