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Review
. 2020 Apr 1;12(4):a036319.
doi: 10.1101/cshperspect.a036319.

BAX, BAK, and BOK: A Coming of Age for the BCL-2 Family Effector Proteins

Tudor Moldoveanu  1   2 , Peter E Czabotar  3   4
Affiliations
Review

BAX, BAK, and BOK: A Coming of Age for the BCL-2 Family Effector Proteins

Tudor Moldoveanu et al. Cold Spring Harb Perspect Biol. .

Abstract

The BCL-2 family of proteins control a key checkpoint in apoptosis, that of mitochondrial outer membrane permeabilization or, simply, mitochondrial poration. The family consists of three subgroups: BH3-only initiators that respond to apoptotic stimuli; antiapoptotic guardians that protect against cell death; and the membrane permeabilizing effectors BAX, BAK, and BOK. On activation, effector proteins are converted from inert monomers into membrane permeabilizing oligomers. For many years, this process has been poorly understood at the molecular level, but a number of recent advances have provided important insights. We review the regulation of these effectors, their activation, subsequent conformational changes, and the ensuing oligomerization events that enable mitochondrial poration, which initiates apoptosis through release of key signaling factors such as cytochrome c We highlight the mysteries that remain in understanding these important proteins in an endeavor to provide a comprehensive picture of where the field currently sits and where it is moving toward.

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Figures

Figure 1.
Figure 1.
The BCL-2 family and the intrinsic pathway to apoptosis. (A) Family members of the BCL-2 protein family. The family is made up of three subgroups of proteins related to each other by regions of sequence homology, the so-called Bcl-2 homology (BH) domains. Regions of secondary structure and domains discussed in the text are labeled. (B) BH3-only proteins, which generally only possess the BH3 domain, are up-regulated on apoptotic stimuli to initiate signaling of the pathway. BH3-only proteins interact with both the effectors BAX and BAK, and the antiapoptotic guardians. Guardians can protect against apoptosis by sequestering both the BH3-only proteins, thus inhibiting effector activation, and by neutralizing activated effector proteins directly. If freed, activated effectors oligomerize at the mitochondrial outer membrane leading to permeabilization of this barrier. This enables the release of apoptogenic factors into the cytosol, primarily cytochrome c (cyt c), leading to caspase activation and ensuing apoptosis. Emerging evidence indicates that BOK is a third member of the effector subgroup with alternative mechanisms of regulation (discussed later).
Figure 2.
Figure 2.
Regulated recruitment of cytosolic BAX to mitochondria. (A) The nuclear magnetic resonance and crystal structures of wild-type (WT) and P168G mutant full-length hBAX (FL BAX) revealed the dormant conformation of cytosolic BAX, with the carboxy-terminal transmembrane (TM) helix α9 bound to and occluding the BAX canonical hydrophobic groove (front view). The same colors have been used in structural figures throughout for different secondary structure elements and regions. PDB identifiers are included (see Table 1 for a summary of effector PDBs). (B) The crystal packing dimer of BAX P168G mutant. The proposed regulatory α1–α2 loop is highly dynamic and found at the rear site. (C) Schematic of retrotranslocation and possible inhibition of mitochondrial BAX by parkin ubiquitylation (Ub). (D) Mitochondrial recruitment is also thought to be regulated by mitochondrial dynamics and by VDAC complex via VDAC2 interactions. Parkin ubiquitylation of VDAC2 blocks BAX recruitment. BAX phosphorylation by AKT at position S184 blocked its mitochondrial association. (E) Chemically stapled BIM, BH3-stabilized α helix of BCL-2 protein (SAHB) has been proposed to displace the regulatory α1–α2 loop at the rear to induce allosteric changes that ultimately destabilized the TM interaction at the front activation groove to drive targeting of BAX to the mitochondria via TM (see D).
Figure 3.
Figure 3.
Structural basis of BAX and BAK direct activation through the canonical BH3 binding groove and selective human BAK inhibition. (A) BH3 peptide complexes with BAX (top) and BAK (bottom), aligned on apoBAK, reveal the overall similar arrangement of BH3 helices at the activation grooves. Six to eight hydrophobic residues of the BH3 peptides engage six hydrophobic pockets (numbered P0–P5). BAX and BAK grooves are occluded in apo forms at P1–P2, and P2–P3, respectively. Chemical stapling induced a slight shift of the P0–P3 portion of the BID BH3 helix at the activation groove of BAK. BH3 binding involves opening of the occluded grooves in BAX and BAK and formation of deep pockets not engaged by peptide residues (van der Walls contacts 4 Å from the nearest peptide atom) (orange). (B) Structure-guided design of BIM BH3-based molecular glue that locks BAK in an inactive conformation similar to apoBAK by introducing stabilizing salt bridges that block helix α1 release. (C) BAX and BAK engage BH3 peptides similarly at the canonical groove yet they show different peptide-induced changes, which manifest by formation of engorged cavities at the regions indicated by the arrows. Destabilized regions in BAX are at the groove region near helix α3 and α8, and in BAK are on either side of the carboxyl terminus of helix α2 and the amino terminus of helix α3. Rotation is relative to the front view. BH3 peptide alignments indicate the position of the hydrophobic residues (H0–H5) that make contact with hydrophobic pockets (P0–P5) in BAX and BAK. H0–H5 residues in BID and BIM (red) were deduced from structures of respective peptides in complex with BAX and BAK. Purple residues are hydrophobic residues found at the putative positions in other BH3 peptides expected to more weakly activate effectors compared with BH3 peptides from BID and BIM. A conserved BH3 aspartic acid (black highlight) forms a stabilizing salt bridge with a conserved arginine in the groove of BAX and BAK (B). A small residue (glycine or alanine, labeled s), allows tight contact between the BH3 helix and the groove next to the conserved salt bridge.
Figure 4.
Figure 4.
Structural transitions during BAX and BAK activation. (A) Activation of monomeric BAX and BAK leads to a series of structural transitions eventuating in dimerization, oligomerization, and ultimately pore formation. Of this sequence, high-resolution structural details are available for monomeric forms of the proteins (Fig. 2A), BH3 activation at the canonical groove (Fig. 3), core from latch detachment (B), and core domain dimers as in C. (B) One of the structurally characterized conformation changes is disengagement of the core domain from the latch region. (C) Once released, core domains dimerize through a symmetric BH3-in-groove protein–protein interface. The dimer produced has a hydrophilic surface dominated by α2–α3 and a hydrophobic surface lined by α4–α5. The hydrophobic surface is thought to engage the outer mitochondrial membrane as a step toward permeabilization. Structures shown throughout are for BAX, but similar transitions have been shown for BAK as indicated in the text.
Figure 5.
Figure 5.
Effector activation and inhibition with molecules other than BH3 peptides. (A) Proposed small-molecule activators (red), inhibitors (green), and allosteric modulators (orange) of BAX and BAK membrane permeabilization, and their possible interaction sites. The postulated inhibitory site of interaction for BCL-2 BH4 SAHB is also indicated. (B) The human cytomegalovirus protein viral mitochondrial-localized inhibitor of apoptosis (vMIA) inhibits BAX through a short helical peptide. (C) Antibody-mediated activation of BAX involves binding of an epitope of the regulatory α1–α2 loop, which unfolds the carboxyl terminus of helix α1. The heavy and light chains of the 3C10 Ab are colored red and pink, respectively.
Figure 6.
Figure 6.
Dormant BOK is intrinsically unstable. Overlay of chicken and human BOK structures reveals three possible arrangements of the regulatory groove region between helices α3–α4. Additionally, human BOK has instability at the α1 helix, through G35, whose mutation to alanine significantly inhibits membrane association and permeabilization, and blocks BAK- and BAX-independent apoptosis.
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