Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides. The distinguishing characteristics of BH3 peptides capable of directly activating Bax were also elucidated. Here we identify specific BH3 peptides capable of "unlatching" Bak and describe structural insights into Bak activation and oligomerization. Crystal structures and crosslinking experiments demonstrate that Bak undergoes a conformational change similar to that of Bax upon activation. A structure of the Bak core domain dimer provides a high-resolution image of this key intermediate in the pore-forming oligomer. Our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to certain BH3 peptides.

NMR studies of interactions between Bax and BH3 domain-containing peptides in the absence and presence of CHAPS.

Activation and oligomerisation of Bax, a key pro-apoptotic Bcl-2 family protein, are key steps in the mitochondrial pathway to apoptosis. The signals for apoptosis are conveyed by the distantly related BH3-only proteins, which use their short BH3 domain, an amphipathic α-helix, to interact with other Bcl-2 family members. Here we report an NMR study of interactions between BaxΔC and BH3 domain-containing peptides in the absence and presence of CHAPS, a zwitterionic detergent. We find for the first time that CHAPS interacts weakly with BaxΔC (fast exchange on the NMR chemical shift timescale), at concentrations below micelle formation and with an estimated Kd in the tens of mM. Direct and relatively strong-interactions (slow exchange on the NMR chemical shift timescale) were also observed for BaxΔC with BaxBH3 (estimated Kd of circa 150µM) or BimBH3 in the absence of CHAPS. The interaction with either peptide alone induced widespread chemical shift perturbations to BaxΔC in solution which implies that BaxΔC might have undergone significant conformation change upon binding the BH3 peptide. However, BaxΔC remained monomeric upon binding either CHAPS or a BH3 peptide alone, but the presence of both provoked it to form a dimer.

Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis.

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.