Structure assembly of Bcl-xL through α5-α5 and α6-α6 interhelix interactions in lipid membranes

Lipid bilayer membrane is the main site where Bcl-x_L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x_L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x_L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x_L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x_L sequestered tBid by direct interaction at physiological pH. If Bcl-x_L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x_L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x_L. Previously, a crystallography study demonstrated that Bcl-x_L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x_L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x_L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x_L protein.     

The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-x_L, control Bax activity. Bcl-x_L recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-x_L. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-x_L membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-x_L, but not that of Bcl-x_L shuttling. Bax retrotranslocation depends on interaction to the Bcl-x_L membrane anchor and interaction between the Bax BH3 domain and the Bcl-x_L hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction.     

A conserved hydrophobic core at Bcl-xL mediates its structural stability and binding affinity with BH3-domain peptide of pro-apoptotic protein

Bcl-2 family proteins regulate apoptosis through their homo- and heterodimerization. By protein sequence analysis and structural comparison, we have identified a conserved hydrophobic core at the BH1 and BH2 domains of Bcl-2 family proteins. The hydrophobic core is stabilized by hydrophobic interactions among the residues of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 in Bcl-x_L. Destabilization of the hydrophobic core can promote the protein unfolding and pore formation in synthetic lipid vesicles. Interestingly, though the hydrophobic core does not participate in binding with BH3 domain of pro-apoptotic proteins, disruption of the hydrophobic core can reduce the affinity of Bcl-x_L with BH3-domain peptide by changing the conformation of Bcl-x_L C-terminal residues that are involved in the peptide interaction. The BH3-domain peptide binding affinity and pore forming propensity of Bcl-x_L were correlated to its death-repressor activity, which provides new information to help study the regulatory mechanism of anti-apoptotic proteins. Meanwhile, as the tryptophans are conserved in the hydrophobic core, in vitro binding assay based on FRET of “Trp → AEDANS” can be devised to screen for new modulators targeting anti-apoptotic proteins as well as “multi-BH domains” pro-apoptotic proteins.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

BimL displacing Bcl-xL promotes Bax translocation during TNFα-induced apoptosis

Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. Bim_L, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim_L activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of Bim_L in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x_L and Bax decreased after TNFα treatment, while that between Bcl-x_L and Bim_L increased. Bax did not co-immunoprecipitate with Bim_L before or after the TNFα treatment. In addition, the increased interaction between Bim_L and Bcl-x_L was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim_L redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.     

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x_L, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x_L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x_L. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x_L. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x_L and Beclin 1.     

tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.     

Evaluation of the BH3-only Protein Puma as a Direct Bak Activator

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as “direct activators” that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (K_D = 26 ± 5 nm) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.     

A New Assay Based on Fluorescence Resonance Energy Transfer to Determine the Binding Affinity of Bcl-xL Inhibitors

We developed a new assay of Bcl-x_L inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-x_L. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-x_L inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.     

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the α-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques—yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis—to elucidate specificity determinants for binding to Bcl-x_Lversus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x_L selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x_L, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x_L-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x_L binders.     

Bcl-2 and Bax Interact via the BH1–3 Groove-BH3 Motif Interface and a Novel Interface Involving the BH4 Motif

The interaction of Bcl-2 family proteins at the mitochondrial outer membrane controls membrane permeability and thereby the apoptotic program. The anti-apoptotic protein Bcl-2 binds to the pro-apoptotic protein Bax to prevent Bax homo-oligomerization required for membrane permeabilization. Here, we used site-specific photocross-linking to map the surfaces of Bax and Bcl-2 that interact in the hetero-complex formed in a Triton X-100 micelle as a membrane surrogate. Heterodimer-specific photoadducts were detected from multiple sites in Bax and Bcl-2. Many of the interaction sites are located in the Bcl-2 homology 3 (BH3) region of Bax and the BH1–3 groove of Bcl-2 that likely form the BH3-BH1–3 groove interface. However, other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Loss-of-function mutations in the BH3 region of Bax and the BH1 region of Bcl-2 disrupted the BH3-BH1–3 interface, as expected. Surprisingly the second interface was also disrupted by these mutations. Similarly, a loss-of-function mutation in the BH4 region of Bcl-2 that forms part of the second interface also disrupted both interfaces. As expected, both kinds of mutation abolished Bcl-2-mediated inhibition of Bax oligomerization in detergent micelles. Therefore, Bcl-2 binds Bax through two interdependent interfaces to inhibit the pro-apoptotic oligomerization of Bax.     

Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion,Bax translocation,and Bax/Bak oligomerization suppressed

Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-x(L)). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochrome c in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes.     

The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane

Under many apoptotic conditions, Bax undergoes conformational rearrangements, leading to its insertion in the mitochondrial outer membrane as a transmembrane oligomer. At the same time, mitochondria undergo fragmentation and activated Bax was reported to localize to fission sites. We studied how lipid composition and membrane curvature regulate Bax activation. When isolated mitochondria were incubated with phospholipase A2, which led to phosphatidylethanolamine and cardiolipin hydrolysis, tBid and Bax insertion were hindered. We thus studied in liposomes how phosphatidylethanolamine, cardiolipin, and its hydrolysis products affect Bax activation. Whereas phosphatidylethanolamine, a lipid with negative curvature, did not affect Bax insertion, it inhibited Bax oligomerization. Conversely, Bax insertion required cardiolipin, and was not blocked by cardiolipin hydrolysis products. These experiments support a direct role for cardiolipin in the recruitment and activation of Bax. To examine if the increase in membrane curvature that accompanies mitochondrial fission participates in Bax activation, we studied how liposome size affects the process, and observed that it was inhibited in small liposomes (相似文献   

Activation of Bax by joint action of tBid and mitochondrial outer membrane: Monte Carlo simulations

The mitochondrial pathway of apoptosis proceeds when molecules, such as cytochrome c, sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane (MOM) permeabilization. Bax, a member of the Bcl-2 protein family, plays a pivotal role in mitochondrion-mediated apoptosis. In response to apoptotic stimuli, Bax integrates into the MOM, where it mediates the release of cytochrome c from the intermembrane space into the cytosol, leading to caspase activation and cell death. The pro-death action of Bax is regulated by interactions with both other prosurvival proteins, such as tBid, and the MOM, but the exact mechanisms remain largely unclear. Here, the mechanisms of integration of Bax into a model membrane mimicking the MOM were studied by Monte Carlo simulations preceded by a computer prediction of the docking of tBid with Bax. A novel model of Bax activation by tBid was predicted by the simulations. In this model, tBid binds to Bax at an interaction site formed by Bax helices α1, α2, α3 and α5 leading, due to interaction of the positively charged N-terminal fragment of tBid with anionic lipid headgroups, to Bax reorientation such that a hydrogen-bonded pair of residues, Asp98 and Ser184, is brought into close proximity with negatively charged lipid headgroups. The interaction with these headgroups destabilizes the hydrogen bond which results in the release of helix α9 from the Bax-binding groove, its insertion into the membrane, followed by insertion into the membrane of the α5–α6 helical hairpin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices α5 and α6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.     

Helix orientations in membrane-associated Bcl-X<Subscript>L</Subscript> determined by <Superscript>15</Superscript>N-solid-state NMR spectroscopy

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x_L is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x_L associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x_L its membrane interactions are weak. In the presence of membranes Bcl-x_L can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x_L hydrophobic binding pocket becomes accessible for protein–protein interactions whilst the structure of the N-terminal region remains intact. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.