Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis

During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the α1 helix are exposed indicating complete dissociation of α1 from α2 in the core and from α6–α8 in the latch. Moreover, disulfide tethering of α1 to α2 or α6 blocks cytochrome c release, suggesting that α1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when α1 is tethered shows that Bid triggers α2 movement, followed by α1 dissociation. However, α2 reaches its final position only after α1 dissociates from the latch. Thus, α1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (α2–α5) and the latch (α6–α8). During apoptosis, Bak undergoes major conformational changes that lead to mitochondrial permeabilization. Here, the authors characterize changes that occur within the Bak N-terminus using a series of antibodies and a novel tethering approach, demonstrating that dissociation of the α1 helix is a key early step in the unfolding of Bak.

B ak and Bax are pro-apoptotic members of the Bcl-2 protein family that controls apoptosis 1,2 . In healthy cells, Bak and Bax exist in inert conformations in which their hydrophobic a5 helices are surrounded by seven or eight amphipathic helices 3,4 . A carboxy (C)-terminal helix, a9, anchors Bak constitutively in the mitochondrial outer membrane (MOM) 5,6 , whereas in Bax, the a9 helix is initially sequestered in the hydrophobic surface groove, rendering Bax largely cytosolic 4,7 . When cells experience sufficient stress, the BH3-only class of Bcl-2 family proteins cause Bak and Bax to undergo a series of conformation changes-collectively referred to as 'activation'-that culminate in dimerization, leading to pore formation in the MOM [8][9][10] . Pore formation releases cytochrome c into the cytosol to initiate caspase activation and dismantle the cell 11 .
Bak activation is triggered by binding of BH3-only proteins (for example, Bid or Bim) to a hydrophobic groove on the Bak surface 11,12 . Two major consequences are exposure of the BH3 domain in a2 (ref. 9) and exposure of epitopes in the Bak amino (N) terminus 10,13 . The N terminus comprises a semi-structured N-segment (aa1-23) 14 and the a1 helix (aa24-50) containing the BH4 domain 15 . Similar conformation changes occur in Bax during its activation [16][17][18] . Another key conformation change identified in Bak and Bax is separation of the latch (a6-a8) from the core (a2-a5) 19,20 . After these large changes in conformation, the proteins form BH3:groove dimers and make significant contact with the outer membrane surface 9,16,19,21,22 .
Elucidating the structures of the fully activated forms of Bak and Bax will provide insight into how these proteins self-associate to form the apoptotic pore. However, the Bak N-terminal epitopes exposed by activation are poorly mapped so it remains unclear whether some or all elements of the N-termini re-position during activation, or whether they are exposed by re-positioning of other elements, for example, the a6-a8 latch domain 19 or by dissociation of Bak from other proteins such as VDAC2 (refs 10,23). To gain insight into the molecular changes occurring at the Bak N terminus during apoptosis, we mapped the epitopes of 11 anti-Bak antibodies using peptide arrays and mutagenesis. We also developed a novel fluorescence-activated cell sorting (FACS) protocol for measuring epitope exposure after disulfide tethering, which allowed us to examine the sequence of Bak conformation changes during activation.
Herein, we show that N-terminal epitopes of Bak are mostly linear, and that many are located within the a1 helix including two that overlap with the BH4 domain of Bak. All epitopes within a1 and the N-segment are conformation specific as their exposure increases upon Bak activation. Intramolecular tethering demonstrated that separation of a1 and a6, and of both from the core, is required for pore formation. In addition, dissociation of a1 from a2 is required for BH3:groove dimer formation. Thus dissociation of a1 from both the core and latch domains is a key event in the activation of Bak.

Results
Most Bak antibodies have linear epitopes. To investigate Bak N-terminal conformation change during apoptosis, we compared the binding properties of 11 anti-Bak antibodies. Table 1 lists known characteristics of the antibodies, together with the key residues involved in their epitopes, as determined below.
Antibodies were first tested by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting to determine if their epitopes were linear (also called continuous or sequential epitopes) or assembled (also called discontinuous or conformational epitopes) 24 . As all antibodies recognized human Bak after SDS-PAGE (Fig. 1a), each epitope was linear.
Epitopes map to various sites in the Bak N terminus. As the Bak epitopes were linear, further mapping was performed using peptide arrays. When applied to an array of overlapping 15-mer peptides from human Bak, all antibodies bound to at least one peptide (Fig. 1b). When applied to a similar array of mouse Bak peptides, the six antibodies that bound mouse Bak on western blots (Fig. 1a) also recognized at least one peptide ( Supplementary Fig. 1).
Epitope mapping by peptide array was consistent with the immunogen sequence and previous mapping data (Table 1), and together the panel of antibodies recognized at least five distinct epitopes in the N terminus (Fig. 1b). Note, the N-segment positions of the 2-14 and 8F8 epitopes suggested by the array were confirmed by western blotting of N-terminally truncated Bak (Fig. 1c).
As seven antibodies (14-36, NT, a23-38, G317-2, Ab-1, Ab-2, 7D10) bound peptides containing a1 residues, we focused on mapping their epitopes further and designed an array of 8-mers with only a single residue offset to define the minimal set of residues required for their binding. As expected, due to their polyclonal nature the three peptide-derived antibodies (14-36, NT and aa23-38) produced complex binding patterns for this array ( Supplementary Fig. 2), while the monoclonal antibodies (G317-2, Ab-1, Ab-2 and 7D10) produced simple binding patterns. G317-2 bound four peptides (Fig. 2a) refining its epitope to 32 EEVFR 36 , and suggesting that E32 and R36 are particularly important for its binding. Ab-1 and Ab-2 bound predominantly to the same peptide 38 YVFYRHQQ 45 , suggesting their epitopes are very similar. The 7D10 epitope begins after a1, with 51 GVAAP 55 the minimal set of residues required for binding. Thus, peptide arrays of increasing resolution defined the minimal linear epitopes of several antibodies to the Bak N terminus.
The BH4 domain in a1 is critical for Bak stability. To analyse the role of a1 in Bak function, we utilized 10 variants of Bak in which residues (E25, A28, V34, F35, R36, S37, Y38, Y41, R42, Q44) were substituted with cysteine (Fig. 2b,c). After stable expression in Bak À / À Bax À / À MEF, most variants were present at levels similar to that of wild-type human Bak (Fig. 2c), indicating that cysteine substitution had not significantly altered protein expression or stability. The exceptions were three variants (V34C, F35C, Y38C) that were present at only low levels (Fig. 2c). Notably, these residues lie in the BH4 domain of Bak (Fig. 2b), the sequence motif present in all multidomain members of the Bcl-2 family, including viral prosurvival proteins 15 . The destabilizing effect of cysteine substitution within the BH4 domain is consistent with the high conservation of this motif 15 and with observations that truncating Bak a1 results in low protein levels after retroviral expression 14 .
The BH4 domain contributes to the G317-2 and Ab-1 epitopes. The cysteine-substituted Bak variants were also used to test whether the residues within a1 epitopes were required for ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7841 antibody binding (Fig. 2d). Among the polyclonal antibodies, both a23-38 and NT bound weakly to A28C (Fig. 2d), indicating that dominant epitopes among their collections of immunoglobulins overlap and that most depend on A28. The epitopes of 14-36 were also sensitive to mutation of A28 (Fig. 2d). Notably, each of the epitopes for the monoclonal antibodies relied on at least some residues within the BH4 domain ( 34 VFRSYV 39 ). G317-2 failed to bind V34C, F35C and R36C, consistent with its epitope being at EEVFR. Ab-1 and Ab-2 failed to bind Y38C, Y41C and R42C, consistent with their epitope being within YVFYRHQQ, and with a recent report that Y38 is critical for Ab-1 binding 25 . Thus, the anti-Bak antibodies tested bound to three distinct sites (residues 28, 34-36 and 37-44) along a1.
'N-terminal exposure' involves complete dissociation of a1. Antibodies generated to the Bak N terminus have often been used to monitor Bak conformation change (activation) during apoptosis 10,13,14 . In particular, Ab-1 has been widely used as a 'conformation-specific' antibody to report 'N-terminal exposure' of Bak during apoptosis 10 . Based on our mapping of the Ab-1 epitope, two residues (Y38 and R42) required for Ab-1 binding are buried in the structure of non-activated Bak ( Supplementary  Fig. 3), arguing that this central region of a1 must become exposed during apoptosis. A similar argument holds for the start of a1, as the NT antibody recognizes only activated Bak 13 and its epitope includes both A28 (Fig. 2d) and T31 25 , which are also buried in non-activated Bak ( Supplementary Fig. 3). The N-segment also undergoes conformation change, as 8F8 binds preferentially to activated Bak 14 . Thus, based on literature reports of three 'conformation-specific' antibodies (8F8, NT, Ab-1) and epitope mapping of those antibodies, at least three distinct sites (11-17, 28-31 and 38-45) in the N terminus become exposed during Bak activation.
To further examine Bak N-terminal exposure during apoptosis, five additional N-terminal antibodies were tested for immunoprecipitation of Bak before and after activation by Bid (Fig. 3). Three cell lines were used to compare recognition of endogenous mouse and human Bak with exogenous human Bak. All five antibodies bound to Bak more efficiently after its activation (Fig. 3). The 14-36 and G317-2 antibodies were most efficient in the conditions used (Fig. 3, compare bound and unbound fractions). The specificity of these additional five antibodies for an active conformation thus confirms that the entire N terminus (that is, the N-segment and a1) dissociates from the remainder of the protein during Bak activation. Furthermore, when monitored by Ab-1 or G317-2 epitope exposure, this conformation change may be considered 'BH4 exposure', much as 4B5 or G23 epitope exposure signifies 'BH3 exposure'.
Dissociation of a1 from a6 is required for pore formation. To better understand how Bak unfolds during activation, we examined whether BH4 exposure occurs before or after BH3 exposure. This was done by disulfide tethering the N terminus to the remainder of Bak at three positions (Fig. 4a). One tether (WT) utilized the native cysteines in human Bak, C14 in the N-segment and C166 in the a6-a7 loop. Two other tethers (Y41C:A79C and A28C:L163C) involved cysteine substitutions at the relevant positions in cysteine-less human Bak (C14S/C166S). Each tether was induced with high efficiency following addition of the oxidizing agent copper phenanthroline (CuPhe), as indicated by faster migration of Bak during non-reducing SDS-PAGE (Fig. 4b). The mutations did not alter Bak function as indicated by cytochrome c in response to Bid (Fig. 4c). However, in contrast to C14S/C166S Bak, when WT or cysteine double mutant Bak was incubated with CuPhe to induce tethers, cytochrome c release no longer occurred in response to Bid, Bim BH3 peptide or heat (Fig. 4c). These data support a recent report that tethering WT Bak blocks pore formation in response to heat 25 , and defines a1 dissociation as required for Bak pore formation.
To assess which Bak conformation changes were hindered by these tethers, epitope exposure was quantified by flow cytometry. While Ab-1 is commonly used for flow cytometry of Bak, the Y41C substitution disrupted Ab-1 binding (Fig. 4d,e). Instead, we used G317-2, which only recognizes activated Bak (Figs 3 and 4d), and whose epitope also overlaps with the BH4 domain (Fig. 2d). After activation by Bid, the expected binding of G317-2 to all human Bak variants (Fig. 4e), as well as mouse Bak (Fig. 4f), was readily detectable by FACS. Thus, G317-2 is a particularly useful alternative to the Ab-1 antibody as it efficiently recognized activated forms of both human and mouse Bak by flow cytometry (and immunoprecipitation).
When a1 and a2 were tethered (Y41C:A79C) before cells being incubated with Bid, G317-2 bound to the same proportion of cells and with the same efficiency as the untethered samples (Fig. 5a,b). Thus, the tethered a1 and a2 helices were able to dissociate as a unit from the rest of the protein to expose the BH4 domain. In contrast, when a1 and a6 were tethered (A28C:L163C), G317-2 did not bind (Fig. 5a,b), indicating that the a1-a6 tether had prevented movement of a1. This is supported by Ab-1 also failing to bind its epitope (Fig. 5d), which is located further away from   Lysates from MEF expressing no Bak (Bax À / À Bak À / À (DKO)), mouse Bak (Bax À / À ) or WT human Bak (in DKO) were analysed by western blot using the indicated antibodies. Blots were re-probed with b-actin to compare loading. Data are representative of two independent experiments. (b) Most Bak epitopes map to peptides from the N terminus. Histograms showing immunoreactivity of Bak antibodies towards biotinylated 15-mer hBak peptides, as determined by enzyme-linked immunosorbent assay. X axis labels indicate residue number or control reaction conditions. Data are mean and s.d. of three independent experiments. (c) The epitopes of antibodies binding in the Bak N-segment are distinct. The 2-14 and 8F8, which bound to peptides corresponding to N-segment residues in b, were tested by western blot for their ability to bind N-terminally truncated or single residue mutants of hBak (as indicated) expressed in DKO MEF. On the basis of loss of signal, residues 1-7 are required for the 2-14 antibody to bind Bak, whereas residues 8-17 are required for the 8F8 antibody to bind Bak (as also shown in ref. 14). Binding by 4B5 is shown as a reference for expression levels of various mutants, since its epitope in the BH3 domain is C-terminal to the N-segment 9 . Data are representative of two independent experiments. the A28C:L163C tether ( Supplementary Fig. 4). Tethering of WT Bak had an intermediate effect on BH4 exposure, as both G317-2 and Ab-1 binding occurred in the majority of Bid-treated cells ( Fig. 5a,b,d,e), but less antibody bound to each cell ( Fig. 5c,f). We interpret the lower mean fluorescence intensity (MFI) to indicate that all Bak molecules changed conformation in response to Bid, but were only partially exposing the G317-2 and Ab-1 epitopes. Thus, the WT tether is less restrictive than the A28C:L163C tether, and allows sufficient a1 dissociation for some antibody to bind. Notably, when Bak was activated by a Bim BH3 peptide or by heating, BH4 exposure was again restricted by the WT and A28C:L163C tethers ( Supplementary Fig. 5), suggesting that a1 dissociation from a6 is a requisite response to all triggers of Bak activation.
Full BH4 exposure requires core-latch dissociation. Recent studies show that activation of both Bak and Bax requires separation of a5 in the core from a6 in the latch 19,20 , raising the possibility that this event occurs before a1 dissociation. For example, the a1-a6 tether in Fig. 5 might be preventing a6 dissociation rather than a1 dissociation. To test the relationship of core-latch separation to a1 dissociation, we utilized the Bak variant V142C/F150C (ref. 20). When treated with CuPhe, V142C in a5 was efficiently tethered to F150C in a6 and the tether prevented cytochrome c release in response to Bid 20 . We now show that when V142C/F150C is tethered, binding of both G317-2 and Ab-1 still occurs in the majority of Bid-treated cells ( Fig. 6a, b), as found for tethered WT Bak (Fig. 5). As binding of Ab-1 would not occur after dissociation of a6-a8 alone ( Supplementary Fig. 3), a1 dissociation must be initiated before or in concert with core-latch separation. In addition, as antibody binding is limited in the presence of the tether (Fig. 6c), complete a1 dissociation requires a6 dissociation from the core. Altogether, these results indicate that movement of both a1 and the a6-containing latch are required for full exposure of the BH4 domain during activation.
In addition, we found that another measure of Bak a1 conformation change was cleavage by the serine protease enterokinase (EK). EK cleaved Bak only after it had been activated by Bid, and generated an B20 kDa membrane-bound fragment ( Supplementary Fig. 6). Moreover, the non-canonical cleavage site was within the BH4 domain in a1 as the cleaved fragment was recognized by Ab-1 but not G317-2 (Fig. 7a). Notably, cleavage was absent when the N terminus was tethered to the latch (WT, A28CL163C) or the latch was tethered to the core (V142CF150C, Fig. 7b), indicating that those tethers had blocked BH4 exposure, consistent with our FACS results. β-Actin Curiously, cleavage was also absent when a1 was tethered to a2 (Y41CA79C, Fig. 7b) despite full exposure of the G317-2 epitope (Figs 4d,e and 5a-c). We suspect the disulfide bond prevents access of EK, but not G317-2, to its target site. These results therefore confirm that movement of both a1and the a6-containing latch are required for full BH4 exposure during Bak activation.
BH3 exposure is initiated in the absence of BH4 exposure. To test whether a2 movement might precede a1 movement, we measured BH3 exposure when a1 was tethered or untethered. It was plausible that BH3 exposure could occur even if a1 was tethered to the protein core due to the length (19 residues) and flexibility of the a1-a2 loop. To capture BH3 exposure, the 4B5 antibody was added during (rather than after) the incubation with Bid, as the BH3 domain is normally only transiently exposed before it binds to another activated Bak molecule 9 . Accordingly, in the untethered samples, 4B5 added during the incubation bound each Bak variant after Bid treatment (Fig. 8a,b). Even in the tethered samples, the 4B5 antibody bound to all three Bak variants (Fig. 8a,b) indicating that BH3 exposure is a very early step in Bak activation. The a1-a6 tether (A28C:L163C) was most informative as it fully blocked BH4 exposure (Fig. 5) but not BH3 exposure (Fig. 8a,b). Thus, when Bid binds to the Bak hydrophobic groove, BH3 exposure is initiated before BH4 exposure.
We also noted that 4B5 binding was limited in tethered WT and A28C/L163C, as shown by lower MFI after tethering (Fig. 8c). This limited 4B5 binding suggests that flexion of the a1-a2 loop is not sufficient for complete BH3 exposure. Instead, a1 dissociation from the a6-a8 latch domain is also required, as suggested by complete BH3 exposure when a1 was tethered to a2 (Y41C:A79C, Fig. 8a-c).
Dimer formation requires dissociation of a1 from a2. We noted that one tether (Y41C:A79C) allowed Bid treatment to expose both the BH4 and BH3 domains (Figs 5a,b and 7a,b) but did not allow cytochrome c release (Fig. 4c). As a1 was still linked to a2, the exposed BH3 domain may have been unable to bind the hydrophobic groove of another activated Bak molecule to form a BH3:groove homodimer. This was confirmed by Blue-Native-PAGE experiments in which tethered Bak Y41C/A79C failed to dimerize following Bid treatment (Fig. 8d). In addition, the BH3 domain remained exposed as shown by 4B5 antibody binding after (rather than during) the Bid incubation (Fig. 8e,f). Thus, Bak a1 must dissociate from a2 for Bak dimerization and pore formation.
In summary, Bak activation by Bid, Bim or heat proceeds via movement of a2 followed by dissociation of a1 from both the a2-a5 core domain and the a6-a8 latch domain. Separation of the latch from core does not occur before a1 dissociation, but may occur in concert as contacts between helices are progressively lost. The resulting exposure of hydrophobic residues in multiple helices may drive further conformation changes that promote Bak oligomerization due to the newly exposed residues engaging in new protein-protein or protein-lipid interactions.

Discussion
To delineate the Bak 'N-terminal conformation change' known for many years to occur during apoptosis, we mapped N-terminal epitopes (summarized in Fig. 9a) and assayed their exposure in combination with disulfide tethering (Figs 4-8). Our data show that during apoptosis, the N-segment and a1 of Bak dissociate entirely from the rest of the protein to expose the BH4 domain and other residues (Fig. 9b), consistent with recent reports that Bak a1 and a6 became 450 Å apart after tBid treatment in LUV 26 and the distance between Bax a1 and a2 varies widely after activation 21 . Disulfide tethering of Bak showed that a1 dissociation from both the core (a2-a5) and the latch (a6-a8) was required for pore formation. Furthermore, our novel approach of combining tethers with FACS analysis of epitope exposure suggested that Bak conformation change initiates with movement of a2, followed by a1 dissociation, and that both events are required for complete BH3 exposure and subsequent dimerization.
The requirement of a1 dissociation for Bak function is consistent with the role of a1, and more specifically the BH4 domain, in stabilizing the non-activated conformation of Bak. The BH4 motif is highly conserved in multidomain Bcl-2 proteins including viral prosurvival proteins 15 . Its role in stabilizing Bak is indicated by the structure of non-activated Bak in which the four hydrophobic residues in the BH4 domain ( 34 VFxxYV 39 ) contact four neighbouring helices (a2, a5, a6, a7) 3 . R42 may also stabilize non-activated Bak, as structurally this residue participates in a hydrogen bond network with a3 (ref. 3).
As a1 makes many contacts with other helices, a1 dissociation may be triggered by disrupting one or more of those contacts. A recent crystal structure of Bim peptide bound to the Bax groove demonstrated a2 movement, and formation of a cavity between helices 2, 5 and 8 (ref. 19). An NMR structure of Bid peptide bound to the Bak groove also indicated perturbations in several helices including a1, a2 and a6 (ref. 12). The present studies show a2 movement as a very early consequence of Bid binding to the Bak groove. Thus, a2/3 movement and cavity formation may individually or in concert trigger a1 dissociation. ARTICLE Dissociation of a1 allows Bak to unfold into three major components: the N terminus, the core (a2-a5) and the latch (a6-a8). This unfolding exposes many hydrophobic residues in a1, a2 and a5-a8 ( Supplementary Fig. 7) that engage in new protein-lipid or protein-protein interactions to porate the MOM. For example, recent structural and biochemical studies indicate that protein-lipid interactions occur as Bak collapses onto the membrane, with a4, a5 and a6 lying in-plane on the MOM surface 19,26 . The amphipathic a7 and a8 may also lie in-plane to protect their hydrophobic and aromatic residues. Protein-protein interaction generates dimers when the exposed BH3 domain in a2 binds to the hydrophobic groove of another Bak molecule 9,22 . In contrast, a1 remains solvent-exposed as all epitopes can be bound by antibodies in immunoprecipitation or intracellular FACS assays, including those that involve hydrophobic regions such as the BH4 domain. Solvent-exposure of the N terminus is consistent with increased susceptibility of activated Bak to proteolysis by proteinase K, trypsin, calpain 11,27-29 and enterokinase (Fig. 7). Thus, unlike the core and latch domains, the N terminus does not participate in forming the apoptotic pore, consistent with the ability of N-terminally truncated Bak or Bax to permeabilize mitochondria 14,30 .
Dissociation of a1 may also be necessary for heterodimerization with the prosurvival proteins Bcl-x L and Mcl-1, as heterodimers form via a BH3:groove interaction 27,31 . Dissociation of a1 before heterodimerization is consistent with exposure of a calpain cleavage site at R42 in a1 in the Bak:Bcl-x L complex 27   Where Bcl-x L has apparently prevented exposure of N-terminal epitopes in Bak or Bax (as measured by immunoprecipitation assays) 17,25,27 , it is likely that Bcl-x L prevented Bak/Bax activation via Mode 1 interactions with BH3-only proteins 27 or that it masked the Bak or Bax epitopes. Three factors hint that a1 may not retain the helical conformation evident in the structure of non-activated Bak 3 . First, all epitopes in a1 appear linear based on their binding to Bak after SDS-PAGE and to the peptide array. Second, calpain cleaves activated Bak at R42/H43 toward the end of a1 (ref. 12), and calpain rarely cleaves in helical regions 33 . Third, if a1 did remain helical, the hydrophobic surface (for example, the BH4 domain) that normally faces the hydrophobic core of Bak might be expected to make further protein-protein or protein-lipid interactions. But instead, all regions remain available for antibody binding.
Like Bak, Bax N-terminal epitopes (for the 6A7, Bax NT and Bax N20 antibodies) become exposed following apoptosis. Moreover, the epitopes are in the region of Bax a1 (residues 13-19, 1-21 and 11-30, respectively) that abuts the a6-a8 latch domain [34][35][36] , and the epitopes of polyclonal Bax N20 may even include residues of the Bax BH4 domain ( 26 LLQGFI 31 ). As Bax N-terminal exposure is akin to that for Bak, a1 dissociation is probably also required for Bax activation and pore formation.
Indeed, tethering the Bax a1-a2 loop to a6 blocked 6A7 exposure and cytochrome c release in response to SAHB Bim peptide 37 . In this case, the binding site was the 'rear pocket' formed by Bax a1/a6, suggesting that a1 dissociation is a key event in Bax and Bak activation regardless of the initiating interaction.
In conclusion, detailed characterization of the epitopes of several commonly used Bak antibodies, together with a novel tethering approach, enabled us to determine that a1 dissociation is a crucial step in Bak (and probably Bax) activation. With the restraining influence of a1 removed, the freed core and latch domains are able to orient in the membrane, enabling dimerization, oligomerization, cytochrome c release and ultimately cell destruction. Thus, interference with a2 movement or a1 dissociation may represent a new strategy for therapeutic intervention in pathologies associated with overactive or inefficient Bak-mediated apoptosis.
Cell culture. Phoenix cells, DU145 and SV40-transformed mouse embryonic fibroblasts derived from Bak À / À Bax À / À mice (DKO MEF) and Bax À / À mice were a gift from Professor David Huang. Note, the DU145 cells were obtained from the Frederick National Laboratory (USA) as part of the NCI-60 panel of cell lines 39 .
Site-directed mutagenesis. Cysteine variants and N-terminal truncations of Bak were generated by cloning the product of three sequential PCRs (see below) into the pMX-IG vector, kindly provided by Professor David Huang. Primers (Supplementary Table 1) were purchased from GeneWorks Pty Ltd.
For each mutation (except the truncations), two initial paired reactions were performed. One reaction contained the relevant internal (mutagenic) forward primer and a Bak reverse primer that includes an XhoI site. The second reaction contained an hBak forward primer that includes an EcoRI site and the matching internal (mutagenic) reverse primer. Each reaction mixture contained 20 ng template DNA, 0.5 mM of each primer and 1 Â Phusion PCR Master Mix (Finnzymes, #F-531S) and were incubated in an MJ Research PTC-200 Thermal Cycler or a Bio-Rad T100 Thermal Cycler. Cycling conditions were: 94°C, 2 min; 30 cycles of (94°C, 35 s; 58°C, 30 s; 72°C, 60 s); 72°C, 5 min.
The two resulting complementary DNA fragments were gel-purified using the Wizard SV Gel and PCR Cleanup kit (Promega, #A9282) and each pair of DNAs Gel-purified reaction products and the pMX-IG vector were digested overnight at 37°C with the EcoRI and XhoI restriction enzymes (Roche, #10703737001 and #10899194001) in SuRE/Cut Buffer H (Roche, #11417991001). Gel-purified digested mutant Bak and vector DNAs (1:3 vector:insert ratio) were incubated overnight at room temperature in 1 Â ligase buffer containing 2U T4 DNA ligase (Promega, #M1801). Ligation products were used to transform electro-competent JM109 cells and colonies grown overnight on LB agar plates supplemented with 100 mg ml À 1 ampicillin. Subclones were picked, grown overnight and their DNA purified using a QIAprep Spin Mini Kit (QIAGEN, #27106) and stored at À 20°C. Positive clones were identified by restriction digestion, confirmed by sequencing and used for retroviral transfection. Peptide scanning arrays. Three sets of N-terminally biotinylated Bak peptides (with SGSG linker sequence) were synthesized by Mimotopes: (i) 21 15-mer, with a five-residue overlap, spanning all residues of mouse Bak; (ii) 21 15-mer, with a fiveresidue overlap, spanning all residues of human Bak; (iii) 39 8-mer, with a oneresidue offset, spanning residues 20-65 of human Bak. Each lyophilized peptide (1-3 mg each) was resuspended in 400 ml 80% dimethylformamide (DMF) and stored at À 80°C. 96-well plates (Nunc Immuno Maxisorp, #442404 or Corning Costar non-treated PVC, #2797) were coated with 5 mg ml À 1 streptavidin (Sigma S-4762) by incubation for 16-24 h at 37°C. Plates were washed four times with PBS-T (0.1% Tween 20 in PBS), blocked by incubating for 1 h at B21°C in PBS-T containing 0.5% (w/v) BSA (Sigma) and again washed four times in PBS-T. Peptides (in DMF) were diluted 1:1,000 with H 2 O and 100 ml each added to wells of prepared plates. After 1 h incubation, with shaking, unbound peptides were removed by washing four times with PBS-T. Primary antibodies were diluted (see Table 1) in PBS-T and 100 ml incubated with each peptide for at 1 h at room temperature on a rocking platform. Plates were washed four times with PBS-T and 100 ml HRP-conjugated secondary antibody, diluted with PBS-T, was added and plates incubated at room temperature for 1 h on a rocking platform. Dilutions of secondary antibodies were as for western blots, except for peptide set (iii) where the anti-rabbit and anti-rat antibodies were diluted 1:10,000. Plates were washed four times with PBS-T and bound secondary antibody was detected by incubating with 100 ml ABTS buffer (1 mM ABTS (2,2 0 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) diammonium salt, Sigma, #A1888), 100 mM citric acid, 0.03% H 2 O 2 ) for 10-45 min and measuring absorbance at 405 nM using a Hidex Chameleon V Multitechnology Platereader.
Bak tethering and activation. MEF were harvested with trypsin, washed with PBS and the outer cell membrane permeabilized by resuspending cells at 1 Â 10 7 ml À 1 in ice-cold MELB buffer (20 mM HEPES/NaOH pH 7.5, 100 mM sucrose, 2.5 mM MgCl 2 , 100 mM KCl, 1 Â complete protease inhibitor (Roche), 4 mg ml À 1 pepstatin A) containing 0.025% digitonin. After 5 min incubation on ice, cell permeabilization was verified by uptake of trypan blue. For cytochrome c release assays and IPs, permeabilized cells were centrifuged (16,200g, 5 min, 4°C) and membrane fractions resuspended in MELB buffer and kept on ice. For enterokinase cleavage assays of Bak, membrane fractions were permeabilized and subsequently resuspended in MELB buffer lacking 1 Â complete protease inhibitor. Intramolecular tethers were induced by disulfide bonding of cysteines using CuPhe diluted 500-fold into the sample. Permeabilized MEF or membrane fractions were incubated with CuPhe at least 5 min on ice. The efficiency of tethering was assessed by western blotting after mixing untreated or CuPhe-treated samples with an equal volume of sample buffer (0. 15  Binding in groove BH3 partly exposed α1, α6 dissociated, BH4 exposed, hydrophobic core & latch residues exposed α1, α2 dissociated, BH3 fully exposed, hydrophobic core & latch residues re-buried BH3 buried in dimer, BH4 remains exposed Y indicates an epitope is partially accessible to antibody, that is, 4B5 in black, or G317-2 or Ab-1 in green. YY indicates an epitope is fully accessible to antibody. Bak activation is initiated by transient binding of BH3-only domains to the hydrophobic groove, provoking movement of a2 and BH3 exposure. Movement of a2 also breaks a1 contact with the a6-a8 latch, leading to exposure of the BH4 domain and hydrophobic and aromatic residues in the core and latch domains. Dissociation of a1 from the latch allows a2 to reach a position where the BH3 domain is fully exposed. Consequently, exposed hydrophobic and aromatic residues in the core and latch become buried in the membrane, or in interactions with other helices to promote formation of symmetric dimers.
To induce Bak activation, untreated or CuPhe-treated samples were incubated with 100 nM caspase-8 cleaved human Bid or 10 mM Bim BH3 peptide for 30 min at 30°C, or for 30 min at 44°C. Activation reactions were stopped by placing samples on ice for at least 5 min.
Cytochrome c release. Following activation of Bak in membrane fractions, the samples were centrifuged at 16,200g, 5 min, 4°C. The resulting supernatant and pellet fractions were each mixed with sample buffer containing 5% (v/v) 2-mercaptoethanol (2Me) and western blotting performed for cytochrome c.
Immunoprecipitation of activated Bak. Following activation of Bak in membrane fractions, samples were solubilized by incubation with 1% digitonin on ice for at least 30 min. Samples were centrifuged (16,200g, 5 min, 4°C) and supernatants precleared by 30 min incubation at 4°C with Protein G Sepharose beads 4 Fast Flow (GE Healthcare Life Sciences), pre-washed with Onyx buffer (20 mM TrisCl pH 7.4, 135 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 10% glycerol, 1 Â complete protease inhibitor, 4 mg ml À 1 pepstatin A). After removing beads by repeated washes in Onyx buffer, lysates were incubated with constant agitation for 2 h at 4°C with 4 mg ml À 1 anti-Bak antibody, followed by incubation for 1 h at 4°C with additional pre-washed Sepharose G beads. Immune complexes were isolated by centrifugation (16,200g, 2 min, 4°C), washed four times with Onyx buffer, resuspended in sample buffer containing 5% (v/v) 2Me and analysed by western blotting.
Doublets and debris were routinely excluded by gating using forward scatter cytometry and side scatter cytometry. To quantify the proportions of cells with exposed BH4 or BH3 domains, a marker was positioned to distinguish negatively and positively staining populations, with untreated cells (grey histograms) used as guides for negative staining. The MFI of cells in positive-staining populations was also compared in cases where the proportions of cells appeared similar but the amount of staining (that is, profile position on x axis) differed in tethered versus untethered cells.
Bak cleavage by EK. Following tethering and/or activation of Bak in membrane fractions, samples were incubated with or without recombinant EK (3 or 4.4 units per 50 ml sample; Merck #69066-3) for 2 h at room temperature. EK was inactivated by the addition of PMSF (4 mM) and cleavage assessed by western blotting after mixing samples with an equal volume of sample buffer containing 5% (v/v) 2Me.
Blue Native-PAGE. Blue Native-PAGE was performed after Bak activation in permeabilized MEF (as described above) using the Invitrogen NativePAGE Novex Bis-Tris Gel System. All samples were incubated 30 min on ice, centrifuged (16,200g, 5 min, 4°C) and the pellets resuspended in Solubilization buffer (20 mM Bis-Tris pH 7.4, 50 mM NaCl, 10% glycerol, 1% digitonin). After at least 30 min on ice, the samples were centrifuged (16,200g, 5 min, 4°C), the supernatants mixed with NativePAGE sample buffer and NativePAGE 5% G-250 sample additive and proteins separated on 4-16% NativePAGE Novex Bis-Tris gels in chambers containing 1 Â NativePAGE anode buffer and 1 Â NativePAGE cathode buffer containing 1 Â NativePAGE cathode additive. After running at 150 V for 30-45 min, the blue cathode buffer was replaced with 1 Â NativePAGE cathode buffer without any cathode additive and gels run for a further 80-95 min. Proteins were transferred to polyvinylidene difluoride membranes at 40 V for 150 min in buffer containing 25 mM Tris, 192 mM glycine, 20% methanol and 0.037% SDS. Membranes were de-stained by B40 min incubation in fixative [10% acetic acid, 45% methanol]. After washing 3 times for 10 min in water, membranes were incubated in blocking solution and proteins detected by Western blotting. To compare sample loading, excess sample was mixed 1:1 with sample buffer containing 5% (v/v) 2Me and analysed by western blotting.
Western blotting. Samples were heated 5 min at 495°C, spun briefly and proteins separated by SDS-PAGE using pre-cast 12% TGX gels (Bio-Rad) and transferred to nitrocellulose membranes at 40 V for 150 min in buffer containing 25 mM Tris, 192 mM glycine and 20% methanol. Note that transfer of proteins to polyvinylidene difluoride rather than nitrocellulose membranes resulted in inferior signals for most Bak antibodies (data not shown). Nonspecific binding of antibodies was blocked by incubation for 30-45 min with 5% nonfat milk powder in TBS (20 mM TrisHCl pH7.6, 137 mM NaCl) with 0.1% Tween 20. Membranes were rinsed with TBS/0.1% Tween 20 and incubated with primary antibodies at room temperature for 1-5 h or at 4°C overnight. The Ab-1, Ab-2 and 4B5 anti-Bak antibodies and anti-cytochrome c antibody were diluted in TBS/0.05% Tween 20, anti-b-actin antibody was diluted in TBS/5% BSA. All other primary antibodies were diluted in blocking solution. Membranes were washed three times (5 min each) in TBS/0.1% Tween 20 and incubated 1-2 h at room temperature with secondary antibody diluted in blocking solution, except for cytochrome c blots where the anti-mouse secondary was also diluted in TBS/0.05% Tween 20. Membranes were washed three times (5 min each) in TBS/0.1% Tween 20, developed with Luminata Forte HRP substrate (Millipore) and bioluminescent signals detected using a ChemiDoc XRS þ System fitted with ImageLab software (Bio-Rad). Uncropped images from Figs 1-4, 7 and 8 are shown in Supplementary  Figs 8-12.
Structural modelling. The inactive Bak structure 2IMS 3 was downloaded from PDB and manipulated using PyMOL (DeLano Scientific LLC). The images were saved as PNG files.