Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion^*

Abstract

Introduction

Members of the Bcl-2 protein family are essential players in the complex regulation of apoptosis (1, 2). They are divided into three subgroups: the anti-apoptotic Bcl-2-like proteins, the pro-apoptotic multidomain proteins (Bax and Bak), and the pro-apoptotic BH3³ -only proteins. To keep programmed cell death under control, Bax activation needs to be strictly regulated, as abnormal cell death is disadvantageous for multicellular organisms.

Following cleavage by caspase-8, the BH3-only protein, Bid, is known to activate Bax (3,–6). Recently the events involved in Bax-Bid interaction were investigated by fluorescent techniques (7). Bax is activated through a cascade of conformational changes from being inactive and cytosolic to an oligomeric, membrane-inserted state. In the mitochondrial outer membrane (8, 9) activated Bax is responsible for cytochrome c release and apoptosis initiation (10). Bax oligomerization has been shown to occur also in vitro by incubation with detergents (10,–14).

The structures of monomeric Bax and Bid were solved by NMR (14,–16). Bax has a globular fold composed of nine α-helices (α1 to α9), with α2 representing the BH3 domain and α5/α6 the hydrophobic hairpin (see Fig. 1A). Similarities in structure and function with the monomeric inactive form of the channel-forming domain of bacterial colicins or diphtheria toxins are evident (14, 17). The BH3-only protein, Bid, shows a similar globular fold but lacks α8 and α9 and has an additional short helix (α1/2) between α1 and α2. The structure of active Bax is still unknown, but helices α5, α6, and α9 are reported to insert into the outer mitochondrial membrane (see Fig. 1C) (18,–21).

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FIGURE 1.

Monomeric Bax and putative models for the activated state. A, ribbon diagram of the NMR structure of Bax in the inactive monomeric form (Protein Data Bank code 1F16 (14)). The color code is shown in C. The two native cysteines (Cys-62 and Cys-126) are represented as ball-and-stick models (Cβ–Cβ average distance, 2.6 nm). B, sketch of the Bak dimer interface with the elongated helix α2/α3 obtained by cross-linking data (23). C, putative model of Bax inserted in the membrane obtained from cysteine scanning analysis (21).

A number of models have been proposed to describe interactions between Bcl-2 proteins. (i) an NMR study reports an activation mechanism based on the interaction of a stabilized helix of the BH3-only protein Bim with α1 and α6 of Bax, initiating conformational changes in Bax (22). (ii) The Bax homolog Bak is reported to homodimerize by an interaction of a fused α2/α3 helix with the α2/α3 helix of a second Bak monomer (Fig. 1B) (23). (iii) A heterodimerization-based survival mechanism has been proposed in which the BH1–3 domains of anti-apoptotic Bcl-2 proteins form a hydrophobic groove to sequester the BH3 domain of pro-apoptotic family members (24,–26).

However, the molecular details of Bax activation by the BH3-only proteins and the structure of oligomeric/membrane-inserted Bax are still unclear. These areas were investigated here using several biophysical techniques such as AFM, cryo-EM, electrophoresis, mass spectrometry and chromatography, and in particular, site-directed spin-labeling EPR techniques. The latter technique introduces a nitroxide radical probe, methanethiosulfonate spin label (MTSSL), into a protein by covalent bonding to cysteines (the resulting unnatural side chain is referred to as R1 herein). The spin label EPR spectra can be analyzed in terms of the available conformational space of the side chain and the water exposure of the radical. Moreover, the oligomer formation can be followed by the appearance of spin-spin interactions between singly labeled Bax monomers (EPR is sensitive to distances in the 1.5–6-nm range for membrane proteins).

In this work, the formation of Bax oligomers was monitored after (i) incubation with detergents, (ii) reconstitution into lipid bilayers, and (iii) insertion into liposomes in the presence of cleaved Bid. We found that similar oligomers were formed in the presence of high detergent concentration and in liposomes. The EPR-derived kinetics of Bid-induced Bax activation and pore formation showed that the interaction between Bid and Bax has a transient nature and that Bid is not a structural element in the oligomer. Distance measurements performed on assembled Bax oligomers gave insights in the structure of active Bax.

EXPERIMENTAL PROCEDURES

Additional details regarding cell culture, protein preparation, and biophysical techniques used are provided under the supplemental “Experimental Procedures.”

RESULTS

Characterization of Spin-labeled Mutants

Bax has two endogenous cysteines (Cys-62 in the center of α2, BH3 domain, and Cys-126 at the end of α5), both accessible for spin labeling. The doubly spin-labeled Bax is for simplicity named Bax_WT-R1. For spin-spin distance determination in oligomeric Bax, if both positions are labeled, intra- and intermonomer distances are present, which are difficult to disentangle. Thus, to investigate Bax oligomerization steps by EPR, a single spin label was placed per monomeric unit, allowing measurement of only intermonomer distances. The single cysteine mutants were engineered by replacing either cysteine (Cys-62 or Cys-126) by alanine (BaxC62-C126A or BaxC62A-C126). The two singly spin-labeled mutants are for simplicity named Bax_C62R1 and Bax_C126R1, respectively.

Mutagenesis and spin labeling were both tolerated in terms of protein folding and ability to form oligomers. Circular dichroism (CD) spectroscopy performed showed a helical content of about 60% for all produced variants (not shown), consistent with previously published data (14, 32, 33). All Bax variants (spin-labeled or not) in buffer solution were predominantly monomeric and eluted in size exclusion chromatography (SEC) at about 1.7 ml with a reproducible small shoulder at 1.5–1.6 ml, suggesting the presence of a minor fraction of dimers in the preparation (Fig. 2A). The shoulder was also present after spin labeling (more than 90% of the protein population was spin-labeled) or if 2 mm dithiothreitol was present (data not shown). Thus, disulfide bridge formation could be excluded as the dimerization trigger. The dimer fraction was shown to increase after storage of Bax for 10 days at 4 °C (Fig. 2A, top panel).

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FIGURE 2.

Oligomerization assays. A, top panel, size exclusion chromatography on Bax_WT and Bax_WT-R1 (dotted and straight lines, respectively) in the soluble (black) and DDM-induced oligomeric (red) forms. Monomeric Bax_WT after prolonged storage (gray) and oligomeric Bax_WT after detergent removal (pink) are also shown. The elution volumes for the monomeric (M), dimeric (D), and oligomeric (O) states are highlighted by vertical dotted lines. Under the experimental conditions used, standard soluble proteins of 20 and 440 kDa are eluting at 1.7 and 1.15 ml, respectively. Middle panel, monomeric Bax_C62R1 (black) and DDM-induced oligomeric Bax_C62R1 and Bax_C126R1 (straight and dashed red lines, respectively). Bottom panel, full-length Bid in aqueous buffer (black) and in the presence of DDM (red). B, upper panel, blue native gel electrophoresis of soluble (sol) and detergent-preincubated Bax samples (OG, octyl glucoside; DM, n-decyl β-d-maltoside). In the left lane, marker proteins of different sizes are shown. On the right, the putative number of monomers in the oligomers is shown. Lower panel, comparison between Bax and Bid in the soluble and DDM-incubated forms.

Monitoring and Characterization of Bax Monomer to Oligomer Transition

Size exclusion chromatography was used to check Bax oligomerization in detergent. In the presence of high concentrations (1–2% (w/v)) of DDM the main elution peak of all Bax variants (spin-labeled or not) shifted from 1.7 to 1.2 ml, indicative of oligomer formation (Fig. 2A, top and middle panels, and Table 1). A high DDM concentration means that at least one complete micelle (∼150 molecules, ∼80 kDa (28, 29)) per Bax monomer is present. Under the same conditions, ferritin (440 kDa) eluted at 1.15 ml. The size of the Bax oligomer is therefore about 20 times larger than the monomer size (20 kDa) strongly indicating that DDM induces Bax oligomerization, even taking into account the detergent contribution. The elution peak of oligomeric Bax was broader than the peak of the monomeric form (Fig. 2A, top panel), indicating a not perfectly homogeneous oligomer size in the sample. By running DDM-embedded samples (50 μl) with a detergent-free buffer on a detergent-free column (2.4 ml), the DDM concentration was gradually reduced, and the elution peak for Bax shifted to 1.4 ml or ∼100 kDa (Fig. 2A, top panel), suggesting that detergent removal induced disassembly of oligomers.

TABLE 1

Data obtained for Bax_WT, Bax_C62, and Bax_C126 by SEC, mass spectrometry, and EPR

ND, not determined; max. abs., maximum absorption; Sol, soluble; Rec, reconstituted.

	BaxWT	BaxC62	BaxC126
Volume of max. abs. in SEC (ml)
Sol	1.71 (1.73)a	1.72 (1.71)	ND (1.71)
DDM	1.21 (1.25)	1.21 (1.19)	ND (1.22)
Protein mass (Da)
	21,183	21,151	21,151
+MTSSL	21,555	21,335	21,335

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	BaxWT	BaxC62			BaxC126
	Sol	Sol	DDM	Rec	Sol	DDM	Rec
Accessibility (EPR) (MHz)
Wex O2	ND	1b	1	1	3	2	2
Wex NiEDDA	ND	22	11	4	30	4	2
Polarity (EPR) (mT)
Azz	ND	3.64	3.59	3.56	3.72	3.57	3.46
Mean of interspin distance distribution (nm)	3.2		2.3	2.4		3–4c	3–4

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^a Volumes shown in parentheses were obtained with spin-labeled samples.

^b Estimated errors are ±10% for the accessibility data and ±0.01 mT for the A_zz values.

^c High detergent to protein ratio.

In the absence of detergent Bid showed an elution profile similar to that of Bax: a peak at 1.7 ml, attributed to the monomer, with a small shoulder possibly indicating dimer formation. The addition of high DDM concentrations shifted the main elution peak to 1.4 ml (∼100 kDa; Fig. 2A, bottom panel), indicating interaction with a detergent micelle. However, the detergent-induced elution shift was much smaller for Bid than for Bax, suggesting that oligomer formation is an intrinsic property of Bax.

Blue native gel electrophoresis was also performed to investigate Bax quaternary structure under different conditions. In the absence of detergent, Bax_WT showed mainly monomers and dimers (Fig. 2B, upper panel). The increased fraction of dimers (compared with SEC) was possibly induced by the hydrophobic Coomassie G-250 dye in the running buffer. Bax preincubated with various highly concentrated detergents such as octyl glucoside, n-decyl β-d-maltoside, or DDM showed a ladder-like distribution of mostly even-numbered complexes from dimers to dodecamers. In the presence of CHAPS, which is reported not to activate Bax (11, 13), a ladder of both odd- and even-numbered oligomers was formed. Hence, in the presence of CHAPS the dimeric units were not as stable as in the presence of n-decyl β-d-maltoside, DDM, or octyl glucoside. A comparable experiment was performed with Bid (Fig. 2B, lower panel). Consistent with the SEC experiments, the size of Bid was less affected by detergent; the largest oligomer found was a tetramer. In summary, this showed that in detergent Bax has an intrinsic ability to form large oligomers composed of an even number of monomers, indicating the dimers as the stable subunits.

These conclusions were confirmed in cross-link experiments. Cross-linking of monomeric Bax_WT produced mainly monomers and dimers, but after preincubation with n-decyl β-d-maltoside, octyl glucoside, or DDM, a ladder-like distribution of mainly even-numbered oligomers was found. The largest clearly visible cross-linked oligomers consisted of 10 monomers (supplemental Fig. 1 and Ref. 32).

Bax oligomer formation was further investigated by AFM in solution (supplemental Fig. 2). AFM images of protein monomers revealed particles of 3 nm mean height and 7 nm mean diameter (supplemental Fig. 2, B and C). Taking into account that the height is precisely imaged, whereas the diameter becomes exaggerated by the tip, the mean particle size is consistent with the NMR structure (3–5-nm particle diameter) (14). The imaged particles have a volume of 77 nm³ (or 39 nm³ if the diameter is decreased by 2 nm because of tip exaggeration). In the presence of DDM, AFM imaging was disturbed by strong interactions between the tip and the detergent, and thus data collection of Bax oligomers was performed after partial removal of DDM. The imaged particles had a mean height and a diameter only a little larger than the monomers (+0.4 and +2 nm, respectively; supplemental Fig. 2, A–C). The volume (144 or 87 nm³ after a 2-nm correction) is in line with a dimer size. However, cross-linking of Bax_WT in the presence of DDM before adsorption to the mica and subsequent detergent removal led to larger particles of 3–4 nm in height and 10–20 nm in diameter. Moreover, a few particles showed a pore-like structure (supplemental Fig. 2, D–F). In summary, our experiments on Bax in detergent indicated that the protein forms large, even-numbered oligomers, but detergent removal induces a disassembly into smaller oligomers as small as dimers.

Changes in R1 Side Chain Mobility Reflect Bax Conformational Changes

Continuous wave EPR performed on spin-labeled protein reveals information on the molecular environment in terms of the accessibility and motional constraints of the covalently attached spin label (34). The doubly spin-labeled wild type (Bax_WT-R1) and the two singly spin-labeled Bax variants (Bax_C62R1 and Bax_C126R1) were analyzed by continuous wave EPR in the water-soluble state as well as in the DDM-induced or liposome-reconstituted oligomeric state (Fig. 3).

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FIGURE 3.

Room temperature continuous wave normalized EPR spectra of Bax_WT-R1, Bax_C62R1, and Bax_C126R1. Upper panel, spectra of monomeric water-soluble (sol) Bax (black) superimposed over DDM-activated Bax (DDM). The asterisks denote a small fraction of unwashed free label in the DDM samples. Lower panel, spectra of Bax reconstituted (rec) in liposomes from E. coli polar lipids (blue). For comparison the spectra in DDM are superimposed as dotted red lines. For Bax_WT-R1 the spectrum of the oligomeric form reconstituted in liposomes formed by bovine heart lipids is also superimposed in gray.

Bax oligomerization produced significant spectral changes, indicating that both spin-labeled side chains are sensitive to the conformational switch of Bax. The spin labels attached at position 62 in the BH3 domain and at position 126 in helix α5 move from a quite dynamic condition, as reflected by narrow EPR lines in the monomeric form, to a motionally more restricted state in the oligomeric form, as indicated by considerable line broadening. For position 126 the spectral effects induced by DDM and liposomes were almost identical, whereas slightly different spectra were observed for position 62, indicating different interactions of the BH3 domains with the micelle or lipid environment.

The accessibility of the spin label toward the bulk water or the lipid phase was monitored with power saturation continuous wave EPR (35, 36) in the presence and absence of the paramagnetic quenchers NiEDDA (highly soluble in the bulk water) and molecular oxygen (highly concentrated in the apolar lipid environment). Water-exposed spin labels are characterized by high NiEDDA accessibility values and lipid-exposed spin labels by high O₂ values, whereas buried spin labels are inaccessible for both.

In the oligomeric state, Bax_C126R1 showed a major decrease in water accessibility. A comparison of oxygen and NiEDDA accessibility values suggested that C126R1 is located at the membrane-water interface (Table 1). In the oligomeric state, C62R1 was shielded from the bulk water, with the liposomes inducing the biggest effect. The concomitant low oxygen accessibility suggests that the R1 side chain at position 62 is involved in tertiary interactions (Table 1). The A_zz parameters (ranging from 3.3 to 3.7 mT going from apolar to polar spin label microenvironments (34)) extracted from low temperature continuous wave EPR spectra (Table 1), as well as the changes in the spectral form, are consistent with this interpretation.

Kinetics of Bax Activation in the Presence of Liposomes

The EPR technique was used to investigate the influence of pH, Ca²⁺, and Bid derivatives on Bax activation in the presence of liposomes at room temperature (20 °C). EPR spectra of Bax_WT-R1 (to monitor both R1 side chains contemporarily) were recorded at increasing incubation times, normalized, and compared with the normalized spectra of the monomeric-inactive and DDM-activated states (Fig. 4). A pronounced difference in the spectral features can be seen in the low field region of the spectra (box in Fig. 4). Therefore, these regions were shown in the superimposed normalized spectra of Fig. 4.

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FIGURE 4.

Effect of different triggering agents on Bax conformation at room temperature (20 °C). The inset shows two superimposed normalized spectra of Bax_WT-R1 in water (black) and in DDM (red). The low field region of the spectra is enclosed by a square. The changes in the low field region of the spectra of Bax_WT-R1 incubated for 4 min (yellow), 150 min (light green), and 16 h (dark green) in the presence of different triggering agents are presented superimposed on the spectra of Bax in the soluble (black) and DDM (red) forms. The triggering agents added to Bax are indicated. Liposomes were formed with ECL and bovine heart lipids (BHL).

Prolonged incubation of Bax_WT-R1 with p15-Bid (also known as tBid) or p15/7-Bid (also known as c₈Bid; data not shown) in the absence of liposomes induced no spectral changes at room temperature. Incubation of Bax_WT-R1 with liposomes formed with ECL extracts or bovine heart lipid extracts induced slight spectral changes after 16 h of incubation, indicative of a slow partial insertion of Bax into the liposomes (Fig. 4). The insertion of Bax into ECL and bovine heart lipid liposomes was markedly accelerated by p15-Bid or p15/7-Bid and slightly accelerated also by full-length Bid (Fig. 4). The spectral shape of activated Bax is the same as that obtained after detergent activation; thus, this spectral form is assumed to be typical for oligomeric Bax_WT-R1. Enhanced Bax insertion into liposomes was induced by decreasing the pH to 5.4 (as reported in Ref. 37) and by increasing the Ca²⁺ concentration (Fig. 4).

The pro-apoptotic protein Bid is a known trigger of Bax activation; however, its precise role as trigger and/or structural component has not yet been clarified. Moreover, Bax activation can also take place in the absence of any BH3-only proteins (38). To understand Bid-induced Bax activation in more detail, we performed time course experiments at 37 °C on mixtures of Bax_WT-R1 with ECL liposomes and various Bid derivatives at varying stoichiometric ratios (Fig. 5).

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FIGURE 5.

Kinetics of Bax conformational changes at 37 °C. Spectra of Bax_WT-R1 were recorded each 43 s at 37 °C with different triggering agents. A, the intensity of the central EPR line versus incubation time (logarithmic scale) is plotted. Upper panel, effect of p15-Bid in water buffer, DDM, ECL liposomes alone, ECL liposomes and p15-Bid, and ECL liposomes and full-length Bid. Lower panel, effect of ECL liposomes and p15/7-Bid at different Bid-to-Bax stoichiometric ratios. The vertical lines highlight the half-times of the spectral changes from the 100 to 0% monomeric species. B, the EPR spectra detected at time zero (100% monomeric Bax, dotted gray lines) and at the end of the incubation time (0% monomeric Bax, colored).

An increase in temperature from 20 to 37 °C enhanced Bax activation. Even in the absence of Bid, Bax_WT-R1 was activated in the presence of ECL liposomes (with a half-time of about 3 h), as indicated by the EPR spectral change (Fig. 5A, upper panel). This indicates an intrinsic ability of Bax to penetrate into the membrane if retention factors are absent. However, in the presence of Bid derivatives, Bax activation was strongly accelerated. In a 1:1 stoichiometric ratio, p15/7-Bid activated Bax with a half-time of 14 min (Fig. 5A, lower panel), consistent with the data obtained with fluorescent probes (7). The reaction half-time was slower (30 min) when p15-Bid was used at similar stoichiometric ratios (Fig. 5A, upper panel). This slower activation might be explained by an observed tendency of p15-Bid to dimerize and precipitate in solution. Full-length Bid was also found to enhance Bax activation (90-min half-time) but to a lesser extent compared with the cleaved variants (Fig. 5A, upper panel). Incubating Bax_WT-R1 with DDM induced a fast switch (<1 min) toward the oligomeric form (Fig. 5A, upper panel), whereas incubation with p15-Bid in the absence of liposomes did not induce changes in the spectral shape (Fig. 5, upper panels).

Decreasing the p15/7-Bid to Bax stoichiometric ratios from 1:1 to 1:10 or 1:50 increased the half-times 2.9- and 5-fold, respectively (Fig. 5A, lower panel). Notably, after prolonged incubation, the EPR spectra were indistinguishable for all conditions (Fig. 5B, lower panel), indicating 100% efficiency of Bax penetration into the liposomes.

SDS-PAGE of the samples used for the kinetic experiments after 24 h at 37 °C showed that Bax_WT-R1 was partially cleaved in liposome-containing samples (Fig. 6A). The cleavage site was found to be in the loop between α1 and α2 (mass spectrometry data; not shown). No indication of cleavage was found if Bax and p15/7-Bid were incubated in the presence of DDM (Fig. 6B). Notably, the conditions used in this work to induce an almost complete insertion of Bax in the presence of triggering agents (3 h at 37 °C) could be shown to lead to only negligible amounts of truncated Bax (Fig. 6B), thus ruling out their relevance in the data analysis.

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FIGURE 6.

SDS-PAGE of Bax-Bid-ECL liposomes mixtures. A, SDS-PAGE of the samples from the kinetic experiments shown in Fig. 5A after a 24-h incubation at 37 °C. lip., liposomes. B, tests on Bax-Bid-ECL liposomes mixtures or Bax-Bid mixtures upon increasing incubation times at 37 °C. An asterisk marks the conditions used for Bax activation in the EPR experiments, with negligible amount of truncated (tr.) Bax. C, SDS-PAGE of Bax-Bid-ECL liposomes mixtures before and after ultracentrifugation (only pellet fractions loaded) with or without alkali (Alk.) treatment. The enlarged gel part (inset) should allow a visual separation of the p15-Bid and the Bax fragment.

Bax Is Able to Form Pores in Liposomes

Three different techniques were used to show that Bax is active and able to form pores in the membrane bilayer under the experimental conditions used. Cryo-EM was performed on ECL liposomes (∼100 nm size; incubated for 3 h at 37 °C) in the absence (Fig. 7A, upper panel) and the presence of Bax_62R1 and p15/7-Bid (Fig. 7A, lower panel). In the presence of Bax_C62R1 and p15/7-Bid, pores with a diameter of ∼50 nm were observed, consistent with recently published data (39). Membrane incorporation was also verified by detecting the release of nitroxide 4-phosphonooxy-TEMPO radicals or horse heart cytochrome c from the liposome lumen after the addition of Bax and Bid derivatives (Fig. 7, B and C). After incubation, the liposomes were pelleted to detect 4-phosphonooxy-TEMPO in both pellet and supernatant fractions by EPR (Fig. 7B). Cytochrome c was detected by SDS-PAGE in the pellet and by light absorption in the supernatant fraction (Fig. 7C).

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FIGURE 7.

Pore formation assays. A, cryo-EM images. Upper panel, nontreated ECL liposomes were perfectly round and had smooth and continuous membranes. Lower panel, after a 2-h incubation with p15/7-Bid and Bax_C62R1, the ECL liposomes exhibited regions of irregularity and pore-like structures with ∼50 nm diameters. B, cytochrome c release from liposomes. Upper panel, percentage of cytochrome c found in the supernatant. Lower panel, SDS-PAGE performed on the pellet. Standard molecular masses are depicted on the left. C, 4-phosphonooxy-TEMPO release from liposomes. Continuous wave EPR spectra are detected in the pellet (left) and in the supernatant (right). The highest signal intensity is expected in the pellet if liposomes are sealed and in the supernatant if they are open. Two control experiments in the absence of proteins and in the presence of DDM showed the expected behavior.

Control experiments with liposomes alone and in the presence of 1% DDM were performed to detect the background level of 4-phosphonooxy-TEMPO and cytochrome c release for 0 and 100% membrane poration, respectively. The impermeability of the liposomes alone was found to be almost unaffected by incubation at 37 °C and subsequent centrifugation, whereas the detergent provoked the disintegration of the liposomes resulting in a 100% release of the molecules from the lumen (Fig. 7, B and C).

In the absence of Bax, both the full-length and the truncated variants of Bid provoked only minor release of 4-phosphonooxy-TEMPO or cytochrome c from the liposome lumen. The presence of Bax in combination with the Bid derivatives allowed release of 4-phosphonooxy-TEMPO and cytochrome c (100 and 70%, respectively). Liposomes treated only with Bax were also partially opened (Fig. 7, B and C), in line with the kinetic data.

Notably, SDS-PAGE showed that after the Bid-induced membrane insertion of Bax, the latter was found mainly in the membrane fraction, whereas the Bid variants stayed predominantly in the supernatant (Fig. 6C). Only a small fraction of cleaved Bid was found to be membrane-inserted if Bax was present (Fig. 6C).

Interspin Distances in Bax Oligomers

When Bax monomers carrying a single spin-labeled side chain form oligomers, dipolar interaction between adjacent spin labels can be monitored by pulse EPR techniques (DEER) if the interspin distance is in the 1.5–6-nm range (40). In the presence of a homogeneous distribution of singly labeled proteins in the sample, the DEER trace will show a simple exponential decay. If dimers or oligomers are formed and the spin labels are within a distance range of 1.5 to 6 nm, a dipolar frequency (proportional to 1/r³) modulates the DEER trace. The dipolar frequency distribution in the DEER traces was analyzed by the software DeerAnalysis 2008 (31) to calculate the distance distribution between the nitroxide groups within the spin label.

The DEER trace of the monomeric Bax_C62R1 and Bax_C126R1 in water solution showed a simple exponential decay, typical of a homogeneous distribution of monomeric species in the sample (Fig. 8). After the addition of DDM to Bax_C62R1, a dipolar oscillation appeared, indicating intermonomer interactions (Fig. 8A). The derived interspin distance was centered at 2.3 nm. This indicates the presence of at least Bax dimers in the presence of detergent. A similar defined peak centered at 2.4 nm was found for both Bax reconstituted into liposomes and on the pelleted ECL liposomes incubated for 3 h at 37 °C with Bax_C62R1 and p15/7-Bid (Fig. 8A). Decreasing the DDM concentration by a factor of 10 led to a small decrease in the main interspin distance (Fig. 8A).

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FIGURE 8.

Interspin distance analysis. DEER analysis was performed on Bax_C62R1 (A), Bax_C126R1 (B), and Bax_WTR1 (C). Left column, experimental intensity-normalized DEER time traces V(t) and exponentially decaying background signals arising from a three-dimensional distribution of remote spins (thin black lines) obtained with DeerAnalysis 2008 (31). Black, monomeric spin-labeled Bax; red, incubated with1% DDM; blue, liposome-reconstituted. The green trace was recorded for the pellet of liposomes incubated with Bax_C62R1 and p15/7-Bid for 3 h. The brown traces are recorded at a low DDM (0.1%) to protein ratio and the gray traces at a high DDM to protein ratio on the sample containing 25% of the spin-labeled Bax_WT-R1. The traces have been vertically displaced for clarity. Middle column, background-corrected experimental data F(t) fit by Tikhonov regularization with an α parameter 100 (thin black lines). Right column, distance distribution P(r) obtained by the fit. The asterisk denotes a possible artifact peak related to background subtraction and noise. a.u., arbitrary units.

A single distance distribution in an oligomeric state suggests that either the oligomers investigated are pure dimers or the second nearest neighbor distance is beyond the sensitivity of the DEER analysis (>5 nm). The second possibility seems to be more likely, as we have already shown that Bax_WT forms oligomers under the experimental conditions used.

Similar EPR experiments were performed on oligomeric Bax_C126R1 (Fig. 8B). At low detergent concentrations a prominent peak centered at 2.3 nm was found, together with a broad distance distribution in the 3–4-nm range. High detergent concentrations as well as reconstitution into liposomes led to the same distance shift toward 3–4 nm, indicating the formation of similar oligomers in the two conditions tested. The DEER data did not allow us to assign 3 and 4 nm to intra- or interdimer distances. However, they clearly show differences between the oligomers at low or high DDM concentrations, with the latter resembling the liposome-embedded state.

Interspin distances were also determined for Bax_WT-R1. The 3.2-nm distance obtained in the monomeric form (Fig. 8C) is consistent with the Cβ_C62-Cβ_C126 distances from the NMR structures (2.4–2.6 nm; Protein Data Bank code 1F16), taking into account the length of the spin label side chain (0.4–0.8 nm, depending on the torsional states of the C–S bonds (41). Analysis of the overall shape of the distance distribution in the presence of DDM points to an invariant 3-nm distance between C62R1 and C126R1 in both the monomeric and oligomeric forms (Fig. 5C). This was confirmed by the distance distribution centered at 3 nm obtained in the sample containing only 25% of the spin-labeled Bax_WT-R1 (Fig. 8C).

DISCUSSION

In this work, we have presented new structural and kinetic details of Bax oligomerization in detergent and in liposomes, which elucidate some interesting properties of the pro-apoptotic mechanism. Bcl-2 proteins, key factors in the regulation of cell life and cell death, are promising targets in cancer therapy (42). Although sequence conservation within this group is low, the folding of their individual domains is very similar (14, 15, 43). The function of Bcl-2 proteins and their interactions are still not fully understood, and different models for dimeric or membrane-inserted conformations are discussed in the literature (21, 23, 44, 45).

High detergent concentrations, liposome reconstitution, or insertion into liposomes in the presence of cleaved Bid variants induced Bax oligomer formation. In the latter case, we also showed membrane poration, and thus Bid and Bax heterologously expressed were functional. Membrane poration was monitored by release of 4-phosphonooxy-TEMPO or horse heart cytochrome c from the liposomal lumen and proven also by cryo-EM (50-nm pores consistent with the findings of Schafer et al. (39)).

The molecular size of active Bax oligomers is still under debate. SEC, used extensively to follow oligomerization in Bax and Bak (8, 13, 23, 32, 46), showed that the effect of the micelle on oligomer size estimation is difficult to quantify. However, comparing Bax and Bid under similar conditions and performing concomitantly blue native PAGE and AFM experiments, we showed that detergent dilution provoked disassembly of Bax oligomers down to dimers and that oligomerization is an intrinsic property of Bax. These results clearly prove that the protein to detergent ratio is crucial for oligomer formation and maintenance, consistent with a recent study (46) performed on a truncated version of Bax (BaxΔC, lacking α9). However, these authors concluded that BaxΔC is mainly monomeric in the presence of detergents or could be at least dimeric in dodecyl maltoside and Triton X-100 (46). We cannot exclude that despite being active, BaxΔC behaves differently than the full-length protein used here, which clearly at least dimerizes (even at low detergent concentration, as proven by EPR) and subsequently forms larger oligomers (at least at high detergent concentration).

In this work, the largest identified oligomers consist of 12 (blue native gels) or 10 (cross-linking in detergents) subunits, consistent with a 240-kDa protein complex. AFM images of cross-linked Bax subunits showed particles with a 10–20-nm diameter. Bax oligomers with 10–12 units are not compatible with the formation of a pure proteinaceous pore with a diameter of 50 nm (cryo-EM data). This poses the question as to whether the pore is formed only by proteins or also by lipids and if the overall topology of the oligomers is the same in micelles and liposomes. The EPR data provided evidence only on the short range topology of the oligomer; the dimeric structure (2.4-nm distance between C62R1 sites) and the intra- and interdimer spacing between neighboring hairpins (3–4 nm among C126R1 sites) are the same in micelles and liposomes. However, the role of the lipids might not be perfectly mimicked by the detergent micelles, and thus the long range topology of the oligomer could be different, allowing for example a higher number of monomers to be allocated in a purely proteinaceous pore in the membrane. On the other side, in the membrane, Bax oligomers as small as those observed in detergent via SEC, cross-linking, and AFM could also initiate the formation of several small pores that induce the breaking of the vesicles observed by cryo-EM.

Furthermore, if lipids are involved in the pore formation, as suggested by experiments with the α5 peptide of Bax incorporated into a lipidic membrane (47, 48), small oligomers could be sufficient to trigger the formation of a lipidic pore. Clearly, further analysis on the nature of the pore is needed to resolve this issue.

Our data confirmed that Bax activation strictly requires the membrane bilayer. In its absence, a permanent conformational change of Bax induced by Bid was not detected. We found a slow Bax insertion into liposomes in the absence of triggering factors at 37 °C and in the presence of full-length Bid, which was not expected, although it has already been shown in vivo that Bax activation is possible in the absence of Bid and Bim (38). To analyze the self-insertion phenomenon, we performed SDS-PAGE of the samples used in the kinetics experiments (incubated for 24 h at 37 °C), which revealed partial truncation of Bax. The mechanism of Bax truncation is not clear as yet. Notably, truncation appears only after prolonged incubations (>3 h) and is not induced by Bid derivatives or by residual proteases co-purified with the proteins. Moreover, truncation was observed in different liposome preparations, with different batches of lipids.

Mass spectrometry performed on the cleaved Bax showed that the cutting site is located in the loop between α1 and α2. Cleavage in this loop region is known to increase Bax activity (49, 50) and induce a BH3-only-protein like behavior (51). Thus, the observed self-insertion of Bax might be explained by its own induced BH3-only protein activity in vitro. The rate of insertion was, however, strongly accelerated at 37 °C in the presence of all Bid variants.

In the presence of p15- or p15/7-Bid (1:1 ratio with Bax) and liposomes, the EPR-detected conformational change of Bax has a half-time of about 30 and 14 min, respectively. This reaction time is consistent with the time range found in the cytochrome c release experiments (Fig. 7B) and published fluorescence resonance energy transfer (FRET)-based kinetics (half-time for insertion and oligomerization, 10–15 min (7)). Interestingly, decreasing the p15/7-Bid to Bax ratio from 1:1 to 1:50 still induced a 100% insertion of Bax, suggesting a catalytic role for p15/7-Bid, e.g. one p15/7-Bid molecule can run several Bax activation cycles. Moreover, this confirmed the transient nature of Bax-Bid interaction and the irrelevant structural role of Bid in the oligomers. This would explain why experiments performed to show this interaction were only partially successful (see for example Refs. 52 and 53).

The question arises as to where most of the Bid molecules are located during and after Bax activation. The p15-Bid fragment was described to be mostly in the membrane fraction (7, 54). In this work, p15/7-Bid was not membrane-inserted after incubation with liposomes in the absence of Bax (3 h, 37 °C). In the presence of Bax only a minor fraction of p15/7-Bid could be found in the pellet, together with most Bax molecules. Treatment of membranes at higher pH did not affect Bax localization, whereas it almost completely removed p15/7-Bid, indicating that p15/7-Bid is only loosely bound to the membrane surface.

The differences between our results and those of Lovell et al. (7) (using p15-Bid instead p15/7-Bid) might be explained by the presence of the p7 fragment in our work, which covers the hydrophobic surface of the p15 fragment (15), thus impairing a strong interaction with the membrane. Additional proteins might stabilize p15-Bid at the membrane in cells (54).

EPR experiments revealed new molecular details of the conformational switch from the Bax inactive monomeric species to the active oligomeric form. On the basis of the EPR accessibility data, side chain 126 in α5 is suggested to be located at the interface between the membrane and the bulk water, whereas side chain 62 is in a protein interface. These findings are in agreement with the model of Bax (21) in which α5, α6, and α9 are inserted into the lipid bilayer as well as with the models of Bid suggesting that α5 lies on the membrane surface (44, 55).

Detailed distance constraints between spin-labeled positions in the active oligomeric state, in particular between the BH3 domains (position 62) and among the loops connecting the hydrophobic α5 and α6 (position 126), allowed us to model three adjacent Bax dimers. Incubation with DDM, reconstitution into liposomes, or activation with cleaved Bid in the presence of liposomes led to a similar 62-62′ interspin distance distributions centered at 2.3 to 2.4 nm, shown to be only marginally affected by detergent dilution. This indicates a stable interaction between two BH3 domains in a dimeric unit. The invariant monomodal distance distribution observed in the active Bax_C62R1 highlights the role of the BH3-mediated dimers in the oligomer stability and cannot be ascribed to a population of pure dimers (SEC, blue native PAGE, and cross-linking data confirmed the presence of oligomers) nor to oligomers with all BH3 domains equally spaced at 2.4 nm. In the latter case, the distances between alternating BH3 domains are expected to be in the 4-nm range depending on the diameter of the active oligomer and should therefore be visible in the DEER traces. Thus, from the DEER analysis, we also deduced that in the active oligomer the BH3-BH3 dimer interfaces must be separated from each other by more than 5 nm.

The interactions involving α5 and α6 are less stable and environment-dependent. At low detergent concentrations the hydrophobic patches of the protein promote the formation of dimers or small oligomers where the BH3-BH3′ interface is still intact but the hydrophobic α5 and α6 helices are closely packed. Only by increasing the detergent concentration or by insertion into liposomes can a native-like oligomer be formed with 126-126′ intra- and interdimer distances in the 3–4-nm range.

The present data are consistent with the symmetric dimer model based on cross-linking studies on the Bax-analogous protein Bak (23) showing that the two molecules share the same activation mechanism. On the other hand, our data exclude a Bax oligomer arrangement similar to the reported swapped Bcl-xl dimers that interact by fused α5/α6 helices lying side by side and forming the dimer interface (45). If oligomeric Bax had a similar conformation, short 126-126′ and long 62-62′ interspin distances would result, which was not observed.

Fig. 9 presents our model of active Bax and the role of Bid derivatives in Bax activation. In solution, Bax and p15/7-Bid interact transiently. In the presence of the membrane bilayer, Bid act as catalytic trigger for Bax membrane insertion and oligomerization. Once Bax is oligomeric and the pore is formed, Bid recruits further Bax molecules from the water solution.

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FIGURE 9.

Model of adjacent Bax dimers. The joint action of p15/7-Bid, the membrane bilayer, and Bax is shown as the triggering event for Bax dimerization and subsequent oligomerization. Three neighboring Bax dimers are inserted in the membrane bilayer according to the measured EPR distance constraints. Helices α2, α5, and α6 are shown as orange, red, and brown cylinders, whereas the other six helices are not represented. A small fraction of the p15/7-Bid moieties are shown bound to the membrane according to the SDS-PAGE data. Upon oligomerization a pore is formed that can release cytochrome c. Inset, schematic representation of the interaction interface between two neighboring Bax monomers. The molecules were created with the program COOT (56) and visualized with PyMOL (58). Two modified Bax moieties were rotated and translated to build up an interaction area in line with the cross-linked sites of Bak (23) and the 62-62′ EPR distance constraints of Bax (∼2.2 nm in the model). On the right, the elongated helices α2/α3 were rotated to create the proposed salt bridge interactions. Color coding of the amino acids is according to the hydropathic index by Kyte and Doolittle (57). Acidic (Glu and Asp) and basic (Lys and Arg) amino acids are shown in red and blue, respectively. Hydrophobic amino acids (Ile, Val, Leu, Ala, Cys, Phe, and Met) are shown in green and slightly hydrophobic residues (Gly, Thr, Ser, and Trp) in light green. The cysteine at position 62 is yellow.

The model shows three adjacent Bax dimers. The membrane topology of the hydrophobic hairpin (of α5 and α6) and of the BH3 domains conforms to the accessibility and polarity data. The BH3 domains of Bax lie on the membrane bilayer and interact with their nearest neighboring BH3 domains, forming a stable dimer interface (62-62′ distance of 2.4 nm). Residue 126 is placed 3 nm away from C62R1 on the other side of the membrane bilayer. The intra- and interdimer distances between the α5 and α6 loops (carrying C126R1) are in the 3–4-nm range.

We modeled the BH3-BH3 interface starting from the NMR structure of Bax (14) and moving α2 to create the elongated helix α2/α3 (supplemental Fig. 3). Two Bax molecules were aligned according to the Bak cross-linking data (23) (supplemental Fig. 4) and the 62-62′ EPR-derived mean distance. This interface could be built either by facing the hydrophobic residues toward each other (Fig. 9, inset, left panel) or with a stable zipper-like salt bridge network (Fig. 9, inset, right panel). Our data support a model in which, after transient activation by Bid, Bax BH3 domains recognize each other, triggering an irreversible dimer formation that is the nucleation event for the oligomerization and subsequent membrane permeabilization. Trace amounts of cleaved Bid molecules are sufficient to trigger complete Bax oligomerization and insertion, thus excluding Bax-Bid hetero-oligomerization for membrane poration.

Supplementary Material

Acknowledgments

REFERENCES