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

Materials

Reagents were obtained from the following suppliers: lipids and extruder from Avanti Polar Lipids, CM5 biosensor chips from GE Healthcare, Polysorbate 20 from Biacore AB, Q-VD-OPh from SM Biochemicals (Anaheim, CA), glutathione from Sigma, glutathione-agarose and bismaleimidohexane (BMH) from Thermo Scientific, S protein-agarose and Ni²⁺-nitrilotriacetic acid-agarose from Novagen, and FITC-labeled dextran 10 (F-d10) from Invitrogen. Antibodies to the following antigens were purchased from the indicated suppliers: heat shock protein 60 (Hsp60), Bax and green fluorescent protein (GFP) from Cell Signaling Technology, cytochrome c from BD Biosciences, Bak from Millipore, and β-actin (goat polyclonal) from Santa Cruz Biotechnology. Anti-S peptide antibody was raised in our laboratory as described (64). BH3 peptides were generated by solid phase synthesis in the Mayo Clinic Proteomics Research Center (Rochester, MN).

Protein Expression and Purification

Plasmids encoding BakΔTM (GenBank^TM BC004431, residues 1–186) in pET29b(+) and pGEX-4T-1 (65) were kind gifts from Qian Liu and Kalle Gehring (McGill University, Montreal, Canada). Plasmids encoding Bak mutants were generated by site-directed mutagenesis. All plasmids were subjected to automated sequencing to verify the described alteration and confirm that no additional mutations were present.

To express Pumaα in Escherichia coli, cDNA encoding full-length Pumaα (GenBank^TM NM_001127242) was cloned into pET29a(+) to yield a construct with an N-terminal S peptide tag and C-terminal His₆ tag. To improve expression in E. coli, rare codons, including codons for proline (CCC) and arginine (AGG, AGA), were mutated to more commonly used synonymous codons by site-directed mutagenesis. The plasmid was subjected to automated sequencing to verify the described alterations.

Plasmids were transformed into E. coli BL21 (DE3) by heat shock. After cells were grown to an optical density of 0.8, isopropyl 1-thio-β-d-galactopyranoside was added to 1 mm, and incubation was continued for 24 h at 16 °C. Bacteria were then washed and sonicated intermittently on ice in TS buffer (150 mm NaCl containing 10 mm Tris-HCl (pH 7.4) and 1 mm PMSF). All further steps were performed at 4 °C.

His₆-tagged proteins were applied to Ni²⁺-nitrilotriacetic acid-agarose. After columns were washed with 20 volumes of TS buffer followed by 10 volumes TS buffer containing 40 mm imidazole, proteins were eluted with TS buffer containing 200 mm imidazole.

GST-tagged proteins were incubated with glutathione-agarose overnight at 4 °C. Beads were then washed twice with 20–25 volumes of TS buffer and eluted with TS containing 20 mm reduced glutathione for 30 min at 4 °C.

Surface Plasmon Resonance (SPR) Analysis

Proteins for SPR analysis were further purified by FPLC on Superdex S200 column, concentrated in a centrifugal concentrator (Centricon, Millipore), dialyzed against Biacore buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 0.05 mm EDTA, and 0.005% (w/v) Polysorbate 20), and stored at 4 °C for <48 h before use.

Puma BH3 peptide or Puma protein was immobilized on a CM5 chip using a Biacore T200 biosensor. Binding assays were performed at 25 °C using Biacore buffer containing GST or GST-BakΔTM (wt or mutant) injected at 30 μl/min for 1 min. Bound protein was allowed to dissociate in Biacore buffer at 30 μl/min for 10 min and then desorbed with 2 m MgCl₂. Binding kinetics were derived using BIAevaluation software (Biacore, Uppsala, Sweden). Similarly, His₆-tagged BakΔTM on a CM5 chip was exposed to Bim BH3, Puma BH3, Bad BH3, and Puma BH3 wild type peptide or mutants.

Preparation of FITC-Dextran Lipid Vesicles

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-plamitoyl-2-oleoyl-sn-glycero-3-phosphoethan-olamine, l-α-phosphatidylinositol, cardiolipin, cholesterol, and 18:1 DGS-nitrilotriacetic acid (Ni²⁺) at a weight ratio of 36:22:9:8:20:5 were dried as thin films in glass test tubes under nitrogen and then under vacuum for 16 h. To encapsulate FITC-labeled dextran 10 (F-d10), 50 mg of lipid in 1 ml of 20 mm HEPES, 150 KCl (pH 7.0) buffer was mixed with 50 mg of F-d10, sonicated, and extruded 15 times through a 100-nm polycarbonate membrane. Untrapped F-d10 was removed by gel filtration on Sephacryl S-300 HR (GE Healthcare). Phosphate was determined by colorimetric assay (Abcam, Cambridge, UK).

Liposome Release Assay (Modified from Oh et al. (66))

Release of F-d10 from large unilamellar vesicles was monitored by fluorescence dequenching using a fluorimetric plate reader. After purified His₆-BakΔTM together with BH3 peptides was added to large unilamellar vesicles (final lipid concentration 10 μg/ml), 96-well plates were incubated at 37 °C with mixing and assayed (excitation 485 nm, emission 538 nm) every 10 s. F-d10 release was quantified by the equation ((F_sample − F_blank)/(F_Triton − F_blank) × 100%), where F_sample, F_blank, and F_Triton are fluorescence of reagent-, buffer-, and 1% Triton-treated large unilamellar vesicles.

BMH Cross-linking Assay

50 nm His₆-BakΔTM together with 200 nm BH3 peptides were incubated with large unilamellar vesicles at 37 °C for 30 min. BMH was then added to a final concentration of 100 μm, and cross-linking was allowed to proceed at 23 °C for 30 min. After the reaction was stopped by incubation with 5 mm DTT for 15 min, samples were diluted in SDS sample buffer, separated on a 12% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-Bak antibody.

Analytical Gel Filtration

Purified His₆-tagged Bak C14S/C166S (20 μm) with or without the indicated BH3 peptide (60 μm) was incubated in CHAPS buffer (2% CHAPS, 150 mm NaCl, 20 mm HEPES (pH 7.5)) at 23 °C for 3 h. After 200-μl samples were injected onto a Superdex S200 size exclusion column, 500-μl fractions were collected, subjected to SDS-PAGE, and blotted with anti-Bak antibody. Molecular markers (Sigma) in the same buffer were also separated on the same column.

Cytochrome c Release

His₆-BakΔTM was dialyzed against mitochondria buffer (150 mm KCl, 5 mm MgCl₂, 1 mm EGTA, 25 mm HEPES (pH 7.5)) and diluted into mitochondria buffer with 5 mm DTT. Mitochondria purified from Bak^−/−Bax^−/− MEFs (67) were incubated with His₆-BakΔTM and the indicated BH3 peptides at 23 °C for 2 h. After centrifugation (10,000 × g, 15 min), supernatants and pellets were analyzed by immunoblotting.

Cell Culture

Jurkat (T cell ALL) from Paul Leibson (Mayo Clinic, Rochester, MN) and SKW 6.4 (Epstein Barr virus-transformed lymphoma) from American Type Culture Collection were maintained at densities below 10⁶ cells/ml in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FCS), 100 units/ml penicillin G, 100 μg/ml streptomycin, and 2 mm glutamine. 293T cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. Bax^−/−Bak^−/− MEFs were grown in DMEM supplemented with 10% FCS, 250 μm l-asparagine, and 55 μm 2-mercaptoethanol. Bak or Bax was stably introduced into Bax^−/−Bak^−/− double knockout MEFs by retroviral transduction. Beginning 24 h after Bax^−/−Bak^−/− MEFs were infected with pBabe-puro(+) encoding wt human Bak or Bax, cells were selected in DMEM (10% FCS) with 2 μg/ml puromycin for 2 weeks. Stable clones were isolated using cloning rings, expanded, and analyzed for Bak expression by immunoblotting.

Mammalian Expression Plasmids and Transient Transfection

cDNAs encoding human Bim_EL (GenBank^TM AF032457), Pumaα (GenBank^TM NM_001127242), Noxa (GenBank^TM NM_021127), and Bad (GenBank^TM AF021792) were cloned into the Xho1 and BamH1 sites of pEGFP-C1 to yield constructs fused at their N termini to EGFP. Empty pEGFP-C1 served as a control. Plasmids encoding S peptide-tagged Bim_EL, Puma, Noxa, and Bad were constructed in pSPN (64) as described previously (68).

Log phase Jurkat cells or SKW 6.4 cells growing in antibiotic-free medium were transiently transfected with the indicated plasmid using a BTX 830 square wave electroporator delivering a single pulse at 240 mV for 10 ms. Cells were incubated for 24 h and analyzed for apoptosis using APC-coupled annexin V as previously described (68, 69).

Circular Dichroism (CD) Spectroscopy and α-Helicity Calculation

BH3 peptides or Bak as a positive control at 0.2 mg/ml were dissolved in HK buffer (20 mm HEPES, 150 mm NaCl (pH 7.0)) or 30% (v/v) 2,2,2-trifluoroethanol in HK buffer. CD spectra were collected at room temperature on a Jasco Spectropolarimeter 810 (Jasco, Inc., Easton, MD) in a 0.1-cm cuvette. Two sequential scans from 250 to 200 nm were recorded, and the background spectrum of the buffer only was subtracted. The k2d algorithm was utilized to calculate the α helical content of the protein.

Bak Complex Model Generation

The Puma·Bak and PumaIG·Bak complexes were generated from the coordinates of the Noxa·Bak complex (62, 70) in which Noxa was mutated to Puma and PumaIG, respectively, using MacPyMOL V1.5.0 (Schrödinger LLC, Portland, OR). The topology and coordinate files of the two complexes were generated by the tLeap module of the AmberTools12 program (University of California, San Francisco, CA). All His, Glu, Asp, and Cys residues of the two complexes were treated as HIP, GLU, ASP, and CYS, respectively, except that His-164^Bak was treated as HID. The energy minimization of the complexes was performed by using the SANDER modules of the AMBER 11 program Version 11 with a dielectric constant of 1.0 and 200 cycles of steepest-descent minimization followed by 300 cycles of conjugate-gradient minimization using the ff12SB force field (71, 72). The Puma·Bak and PumaIG·Bak complexes were solvated by using the tLeap module with 6,514 and 6,577 TIP3P water molecules leading to a system of 22,548 and 22,736 atoms, respectively, using the distance parameter of 8.2 Å for the SolvateBox command.

Multiple Molecular Dynamics Simulations of the Bak Complexes

Each of the two solvated complexes was energy-minimized for 100 cycles of steepest-descent minimization followed by 900 cycles of conjugate-gradient minimization to remove close van der Waals contacts in the system, then heated from 0 to 300 K at a rate of 10 K/ps under constant temperature and volume, and finally simulated independently with a unique seed number for initial velocities at 300 K under constant temperature and pressure using the PMEMD module of the AMBER 11 program (University of California, San Francisco) with the ff12SB force field (71, 72). Ten 10-ns independent simulations were performed for each of the two complexes. All simulations used (i) a dielectric constant of 1.0, (ii) the Berendsen coupling algorithm (73), (iii) a periodic boundary condition at a constant temperature of 300 K and a constant pressure of 1 atm with isotropic molecule-based scaling, (iv) the Particle Mesh Ewald method to calculate long-range electrostatic interactions (74), (v) a time step of 1.0 fs, (vi) the SHAKE-bond-length constraints applied to all the bonds involving the H atom, (vii) saving the image closest to the middle of the “primary box” to the restart and trajectory files, (viii) a formatted restart file, and (ix) default values of all other inputs of the PMEMD module. All simulations were performed on a cluster of Apple Mac Pros with 1200 Intel Xeon cores (2.4/2.9 GHz).

Bak Complex Model Analysis

Average conformations of the two complexes were obtained by using trajectories saved at 1-ps intervals during the last 2-ns period of the 10 10-ns simulations and the Ptraj module of the AmberTools12 program. These conformations were then energy-minimized using the SANDER module of the AMBER 11 program with a dielectric constant of 40.0 and 100 cycles of steepest-descent minimization followed by 100 cycles of conjugate-gradient minimization. The interaction energy between the BH3 domain and Bak in the energy-minimized average conformations was then calculated using an in-house program with a dielectric constant of 40.0 and a nonbonded cutoff of 50,000 Å.

Graphics

All figures were generated by using the MacPyMOL (V1.5.0) and Adobe Photoshop CS5 Extended Version 12.1 × 64 (Adobe Systems Incorporated, San Jose, CA) programs.

Puma BH3 Binds Directly to Bak

Previous studies examining whether Puma is a direct activator have yielded contradictory results. Accordingly, using recently described approaches (62), we set out to determine whether the Puma BH3 domain (Fig. 1A) interacts directly with the multidomain pro-apoptotic protein Bak. The Bim BH3 domain (Fig. 1A), which has a similar degree of α-helical structure as assessed by circular dichroism spectroscopy (supplemental Fig. S1), served as a control.

When immobilized Puma BH3 peptide was exposed to increasing concentrations of purified, recombinant human Bak lacking the transmembrane domain (BakΔTM), increased binding was detected (Fig. 1B). Likewise, when immobilized BakΔTM was exposed to increasing concentrations of Puma BH3 peptide, increased binding was observed (supplemental Fig. S2A). Direct comparison of Bim BH3 and Puma BH3 binding to immobilized BakΔTM revealed that the Puma BH3 bound to Bak slightly more rapidly but also dissociated somewhat more rapidly (Fig. 1C). Consistent with these results, equilibrium dissociation constants (K_D values) of 260 ± 90 and 290 ± 130 nm were calculated for Bim BH3 and Puma BH3, respectively, binding to Bak (Table 1). In contrast, the Bad BH3 domain barely bound at all (Fig. 1C and Table 1). The addition of CHAPS to the binding assay (62) markedly diminished the dissociation rate (Fig. 1D), as was also seen with the Bim BH3 domain. Accordingly, the K_D values for Puma and Bim BH3 domains bound to Bak decreased to 26 ± 5 and 29 ± 5 nm, respectively, in the presence of 1% CHAPS (Table 1).

To rule out the possibility that binding was unique to Puma BH3 peptides and would not be seen with the Puma protein, we optimized codon usage in the human Pumaα cDNA (supplemental Fig. S3) for expression in E. coli. As was seen with the Puma BH3 domain peptide, Bak bound to the immobilized recombinant Pumaα (Fig. 1E) with a K_D of 140 ± 40 nm. Once again, the addition of CHAPS enhanced the affinity almost 10-fold (Fig. 1F and Table 1).

Puma BH3 Peptide Binds the Canonical BH3 Binding Groove of Bak

Examination of a Puma BH3 peptide in which the three conserved hydrophobic residues in the BH3 domain were changed to glutamate (Puma3E BH3, Fig. 2A) revealed diminished binding to Bak (Fig. 2B and supplemental Fig. S2), just as was previously observed for Noxa3E binding to Bak (62). These observations indicate that residues previously implicated in binding of Noxa to Bak are also important in binding of Puma to Bak.

Recent studies of BH3 peptide binding to Bax have yielded contradictory results, with the Bim BH3 peptide reportedly binding to a shallow groove near the Bax N terminus (75) and alternatively to the canonical BH3 binding groove vacated by the Bax α9 helix after a putative conformational change in Bax (63). To assess whether the Puma BH3 peptide was binding to a portion of Bak corresponding to the so-called “trigger” site on Bax, Arg-36 of Bak was mutated to Ala (Fig. 2C) to mimic the critical K21A mutation in Bax (75). This mutation had no effect on binding of Bak to immobilized Puma (Fig. 2D). In contrast, mutation of critical residues in the canonical BH3 binding groove of Bak (62) diminished the binding of Bak to immobilized Puma BH3 domain (Fig. 2, E and F). These included the Bak R127A mutation, which replaces the invariant Arg presence in the BH3 binding groove of all multidomain Bcl-2 family members (Fig. 2E), and the Bak G126S mutation, which replaces Gly-126 with a bulkier Ser residue that sterically hinders binding of BH3 domains (76). The marked inhibitory effect of these mutations (Fig. 2F) suggests that the Puma BH3 domain binds directly to the canonical BH3 binding groove of Bak and not an alternative site.

Puma BH3 Induces Bak Oligomerization and Bak-mediated Membrane Permeabilization

Our previous results indicated that binding of BH3-only proteins to the BH3 binding groove of Bak is the first step in Bak oligomerization (62). To determine whether the Puma BH3 domain also induced Bak oligomerization, Bak was incubated with liposomes composed of mitochondrial outer membrane lipids (23, 66) in the absence or presence of the Puma BH3 domain peptide, cross-linked with BMH, subjected to SDS-PAGE, and analyzed for dimers by immunoblotting. Positive and negative controls, respectively, were the Bim BH3 peptide, which is known to activate Bak, and the Bad BH3 peptide, which is not able to activate Bak (62). As indicated in Fig. 3A, the Puma BH3 domain, like the Bim BH3 domain, induced Bak oligomerization. In contrast, the Puma3E BH3 mutant, which is unable to bind Bak (Fig. 2B), did not induce Bak oligomerization.

In further experiments Bak oligomerization was also examined by FPLC. In these experiments, the Bim BH3 peptide induced a shift of Bak from monomer to higher order oligomers (Fig. 3B). Likewise, the Puma BH3 domain induced oligomerization, albeit less extensive than that seen with the Bim BH3 domain (cf. two middle panels in Fig. 3B).

To assess whether the oligomerized Bak was biologically active, liposome permeabilization was assessed. The Puma BH3 peptide, like the Bim or Bak BH3 domains, increased Bak-mediated release of fluoresceinated dextran from the liposomes (Fig. 3, C and D, and supplemental Fig. S4). In contrast, like the Bad BH3 domain, the Puma3E BH3 did not (Fig. 3, C and D, and supplemental Fig. S4). Importantly, the Puma or Bim BH3 peptides by themselves had no effect on liposome permeability (Fig. 3, C and D). Collectively, the results in Figs. 2 and 3 indicate that binding of the Puma BH3 domain to the Bak BH3 binding groove is sufficient to induce Bak oligomerization and activation.

Direct Puma·Bak Interactions Are Sufficient for Puma-mediated Killing

Consistent with previous results indicating that transfection of Pumaα is sufficient to induce apoptosis in colon cancer cells and mouse embryonic fibroblasts (27, 52), we observed that EGFP-Pumaα, like EGFP-Bim_EL or EGFP-Noxa, is able to induce apoptosis in SKW6.4 cells (Fig. 4, A–C, and supplemental Fig. S5). In contrast, EGFP-Bad induced much less apoptosis. Likewise, in Jurkat cells, where virtually all mitochondrial apoptosis is Bak-dependent (78, 79), EGFP-Pumaα was readily able to induce apoptosis, as was EGFP-Bim_EL or EGFP-Noxa but not EGFP-Bad (Fig. 4, D–F). Importantly, the ability of EGFP-Pumaα to induce apoptosis in all of these cellular contexts was markedly diminished by the L141E/M144E/L148E mutation in its BH3 domain (Fig. 4G and supplemental Fig. S5).

In further experiments, the ability of Pumaα to induce apoptosis was compared in wild type MEFs, Bax^−/−/Bak^−/− double knock-out MEFs and double knock-out MEFs reconstituted with Bak or Bax. Results of this analysis demonstrated that Pumaα, like Bim_EL and Noxa, was unable to induce apoptosis in the absence of Bax and Bak (Fig. 5A). When MEFs were reconstituted with either Bak (Fig. 5B) or Bax (Fig. 5C), Pumaα was able to induce apoptosis, consistent with a recent report suggesting that Puma can also directly activate Bax (58). Bim_EL likewise induced apoptosis in MEFs reconstituted with either Bax or Bak, whereas Noxa selectively induced apoptosis only in MEFs reconstituted with Bak (Fig. 5, B and C).

A Puma BH3 Mutant That Enhances Puma Function

Close examination indicated that the Puma BH3 peptide bound Bak slightly less tightly (Fig. 1C and Table 1), oligomerized Bak somewhat less effectively (Fig. 3, A and B), and permeabilized liposomes less efficiently (Fig. 3, C and D) than the corresponding Bim peptide. Likewise, EGFP-Pumaα killed SKW6.4 cells less effectively than EGFP-Bim_EL (Fig. 4B). Comparison of the Puma BH3 peptide to other direct activator BH3 domains indicated that the Ile-Gly dipeptide found between the conserved Arg-154 and Asp-157 in Bim_EL is replaced by the bulkier Met-Ala in Puma (Fig. 6A). Molecular dynamics simulations predicted that mutation of Met-Ala to Ile-Gly would make the Puma mutant bind ∼0.5 Å deeper in the Bak BH3 binding groove than the wild type (Fig. 6, B and C), which would also decrease the intermolecular interaction energy (Table 2). In addition, mutation of Met to Ile would allow more spin of the phenyl ring of Phe-93 in Bak, as is evident from the more contracted ring of Phe-93 in the time-average structure of PumaIG·Bak than that in the wild type complex shown in Fig. 6C. Collectively, these observations predict an increased affinity of the PumaIG·Bak complex that is due to improvements from enthalpy and configurational entropy (77).

To test these predictions, we compared the Puma and PumaIG BH3 peptides in the assays shown in Figs. 1–3. Compared with the wild type sequence, the degree of α-helicity of the peptide was unchanged (supplemental Fig. S6). Nonetheless, PumaIG BH3 bound immobilized Bak more tightly (Fig. 7A), oligomerized Bak more effectively (Figs. 3B and 7B), and induced greater Bak-mediated permeabilization of liposomes composed of mitochondrial outer membrane lipids (Fig. 7, C and D). Similar effects on Bak-mediated permeabilization were also observed using purified mitochondria (Fig. 7E). Consistent with these results, Pumaα with the corresponding IG mutation in its BH3 domain also killed SKW6.4 cells slightly more effectively despite somewhat lower overall expression (Fig. 7, F and G).

Conclusions

Previous studies have reached conflicting conclusions regarding the role of Puma in activating Bax and/or Bak. Even in those studies where a direct interaction between Puma and Bax or Bak was suggested, the strength of the interaction was not assessed, and domains responsible for the interaction were not conclusively identified. Recent studies have demonstrated that direct activator BH3 proteins can be distinguished from sensitizers based on (i) their ability to directly bind to Bak and/or Bax by surface plasmon resonance analysis, (ii) their ability to oligomerize Bax or Bak in vitro in the absence of other proteins, and (iii) their ability to induce Bax- or Bak-dependent permeabilization of liposomes composed of mitochondrial outer membrane lipids. Our results demonstrate that under various conditions the Puma BH3 domain binds Bak almost as tightly as the BH3 domain of Bim, a known direct activator. In contrast to results published examining binding of Puma BH3 derivatives to Bax while this work was under review (58), the binding of the Puma BH3 to Bak was observed without introduction of hydrophobic moieties that constrain the conformation of the Puma BH3 domain. Moreover, full-length Pumaα protein retains the ability to bind Bak. This binding involves the canonical BH3 binding pocket of Bak rather than a trigger site on the opposite side of the protein. Mutations that diminish (Puma3E BH3) or enhance (PumaIG BH3) the Puma BH3 domain·Bak BH3 binding groove interaction result in a corresponding change in the oligomerization of Bak, the induction of Bak-mediated liposome or mitochondrial permeabilization, and Puma-induced killing in intact cells. Accordingly, it appears that Puma displays all of the properties of a direct Bak activator.