Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers

Abstract

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

Keywords: Apoptosis; Bak; Bax; Bcl-2; Cell Death; Electron Paramagnetic Resonance (EPR); Mitochondria; Oligomerization Interface; Spin Labeling.

FIGURE 1.

Oligomeric pore formation by BAX (or BAK) and spin labeling of BAK cysteine mutant proteins and their membrane-permeabilizing activity. A, oligomeric pore formation by BAX (or BAK). BAX (or BAK) monomers (41) are hypothesized to form oligomeric pores in the mitochondrial outer membrane, first forming the BH3-in-groove homodimer (6) on the membrane surface, in which helices α2-α5 are highlighted in green and blue. B, sequence alignment of BAX and BAK proteins and the locations of site-directed spin labeling. The residue locations selected for cysteine substitution mutagenesis and spin labeling reaction or chemical cross-linking experiments in mouse BAK (mBAK, middle lines) are marked with colored dots above the amino acid sequences, which are aligned against human BAK and BAX proteins. The yellow and red dots represent the sites selected for spin labeling BAK singly and doubly, respectively. The cyan dots represent sites used for cross-linking experiments. Sequence similarities between the three proteins are rated with a star, colon, dot, and no sign (from the highest to lowest similarity). The α-helices and BH1–3 domains are indicated. This figure was adapted from Oh et al. (13). C, site-directed spin labeling (SDSL) reaction. The MTSSL reacts with the cysteine to form a spin-labeled cysteine residue designated as R1. D, spin labeling efficiency. The average percentage of spin labeling per cysteine residue is shown with the error ranges for each mutant from two experiments. The protein concentrations for certain mutants, e.g. 135R1/164R1, determined by the Bradford assay, might have been underestimated due to cysteine mutagenesis and/or spin labeling, resulting in % labeling efficiency and relative percent release (see Fig. 1E) values greater than 100%. E, the relative percent release of fluorescein isothiocyanate (FITC)-dextran (10 kDa) by the spin-labeled sBAK-ΔC-His proteins before correction for unlabeled cysteine mutant proteins. Liposome dye release experiments were carried out with the indicated spin-labeled sBAK/C154S-ΔC-His proteins (5 nm) in the presence of 25 nm N-terminally His-tagged p7/p15 Bid as described under “Experimental Procedures” (13). The error range is from duplicate experiments (except for 99R1/146R1). F, the relative percent release of FITC-dextran (10 kDa) by the spin-labeled sBAK-ΔC-His proteins after correction for unlabeled cysteine proteins. The contribution of the activity by the unlabeled proteins was corrected for each spin-labeled mutant protein as described under “Experimental Procedures.” G, the relative percent release of FITC-dextran (10 kDa) by the cysteine substitution sBAK-ΔC-His proteins without spin labeling. The dye release activity of the cysteine substitution sBAK-ΔC-His proteins was determined as described in E. All the ribbon diagrams were generated in PyMOL (42).

FIGURE 2.

Distance between two spin labels in the doubly spin-labeled BAK mutants in solution and membrane-inserted states. A, CW EPR spectra of spin-labeled pairs in BAK in solution or in the membrane inserted state. The EPR spectra from the indicated doubly spin-labeled proteins, shown in blue (in solution) and red (in membrane) traces, were normalized to a unit area. The spectral sums of the normalized EPR spectra for the individual R1s in each pair are shown in green (for details, see “Continuous Wave (CW) EPR Spectroscopy” under “Experimental Procedures”). The simulated spectra calculated from the CW deconvolution method for the double mutants (23), showing large line broadening due to the close proximity of the spin labels, are in dotted gray traces. The corresponding distance distribution functions are shown in blue (in solution) and red (in membrane) traces in B (right column) along with the data obtained from the DEER experiments. B, DEER signals and distance distribution probability functions for the R1 pairs in BAK in solution or in the membrane-inserted state. The background-subtracted DEER signals are shown for the BAK mutants with spin labels attached at the indicated positions in solution (blue traces in the graphs on the left column) or in the membrane-inserted state (red traces in the graphs on the left column). Black dotted lines associated with each DEER signals represent the fitted DEER signals. The distance distribution probability functions, P(r), for the corresponding samples obtained either by the Tikhonov regularization or by Gaussian fit are also shown in blue and red traces, for solution and membrane-inserted states, respectively, in the right column. C, schematic representation of conformational changes occurring in BAK upon oligomeric pore formation in membrane. Red dots represent R1 residues. Helices α1 and α6 (and thus α7-α8 as well) move away from helices α2-α5, which appear to be bundled together. Helix α9 is not present in the soluble BAK.

FIGURE 3.

Detection of the intramolecular spin-spin interactions by the DEER (or CW) experiment. A, rationale for the intra-molecular spin-spin interaction by DEER measurement. Left panel, in the presence of 6-fold excess of unlabeled BAK (sBAK/C154S-ΔC-His) protein (black dots), doubly labeled BAK protein (red dot) forms pores in the membrane by the activation with p7/p15 Bid. Only the intramolecular spin-spin interactions between the two nitroxide spin labels (XR1-YR1) will be observed by the DEER approach because the distance between the doubly labeled BAK proteins increase beyond the detection limit. Right panel, a mixture of two singly spin-labeled BAK proteins (green dots) is used in the presence of unlabeled BAK (sBAK/C154S-ΔC-His) at the indicated ratio to form pores. DEER modulations will not be observed in the mixture of two singly labeled proteins due to an increase in the inter-spin distance by dilution with the unlabeled proteins. A CW experiment can also be performed this way. B, DEER data for the doubly spin-labeled proteins and a mixture of two singly labeled proteins in the presence of excess unlabeled proteins. The DEER modulation curves for the indicated membrane-inserted BAK samples prepared with the doubly labeled BAK or the mixture of two singly labeled proteins are shown in red and green traces, respectively. Their two-dimensional (left panels) or three-dimensional (right panels) background signals fitted are shown in black solid lines (for the doubly labeled sample) or black dotted lines (for the mixture of two singly labeled BAK).

FIGURE 4.

Sites of site-directed spin labeling for inter-residue distance measurement between two neighboring BAK proteins in membrane-inserted state. A, sites of spin labeling in BAK. The α-carbon atoms of the 18 residues selected for cysteine substitution mutagenesis and spin labeling reaction are shown in black spheres in a ribbon diagram of the solution structure of sBAK/C154S-ΔC-His. The ribbon diagrams were generated in PyMOL (42) using the coordinates of the mouse BAK obtained by homology modeling after the human counterpart (13). Residues 180–184 are not shown. B, spin labeling efficiency. The percentage of spin labeling per cysteine residue (i.e. per thiol) in each protein was determined (see “Experimental Procedures”). The average values of two measurements are shown with the error ranges indicated. C, the relative percent release of fluorescein isothiocyanate (FITC)-dextran (10 kDa) by the spin-labeled sBAK-ΔC-His proteins after correction for the unlabeled proteins. Liposome dye release experiments were carried out with the indicated spin-labeled sBAK-ΔC-His proteins (5 nm) in the presence of 25 nm N-terminally His-tagged p7/p15 Bid using liposomes (10 μg/ml) encapsulating FITC-dextran (10 kDa) as described (13). The same experiments were carried out with unlabeled cysteine substitution mutants. The corrected activity values for the spin-labeled mutants were calculated as described under “Experimental Procedures.” The average values of two measurements are shown with the error ranges indicated. The protein concentrations for certain mutants, e.g. 135R1, 137R1, 149R1, and 163R1, determined by the Bradford assay, might have been underestimated due to cysteine mutagenesis and/or spin labeling, resulting in % labeling efficiency (Fig. 4B) and/or relative percent release values (Fig. 4C) greater than 100%.

FIGURE 5.

Proximity of helix α5 residues in membrane-inserted BAK oligomers. A, spin dilution experiment for helix α5 residues. The membrane-inserted BAK samples were prepared with the indicated spin-labeled proteins in the presence (3:4) or absence (7:0) of sBAK/C154S-ΔC-His at the indicated ratio as described under “Experimental Procedures.” The area-normalized EPR spectra of the spin-diluted samples (3:4 mixture, black trace) of the indicated membrane-inserted BAK are superimposed to those of the undiluted samples (7:0 mixture, red trace). The ratios of the amplitude of the central line for the 7:0 mixture (h₀) to that for 3:4 mixture (h_d) are indicated. The red arrows for 128R1 indicate the splitting of the EPR lines in the absence of the spin dilution (7:0 mixture) due to the strong spin-spin interactions between two 128R1 residues in close proximity in the membrane-inserted state of BAK (see also Fig. 5C). The hyperfine extrema of the spectrum for the spin-diluted sample (3:4 mixture) are denoted by “im,” which indicate a severely restricted tumbling motion (i.e. immobile) in this spin label. B, distances of 124R1, 142R1, and 143R1 in membrane-inserted BAK to their nearest neighbors estimated by the CW deconvolution method. The CW deconvolution method (23) was applied to the spectra for the indicated mutants (red traces, left panel), resulting in simulated fits (black dotted lines on the left panel) superimposed to the spectra for 7:0 mixture of the indicated residues. The corresponding distance distribution functions are shown on the right panel. C, direct spectral simulation of perdeuterated spin label R1-d15 at residue 128 (128R1-d15) for inter-spin distance estimation. The EPR spectra of the BAK Cys-128 mutant labeled with a perdeuterated spin label (R1-d15, top panel) were obtained in the presence or absence of sBAK/C154S-ΔC-His in membrane at the indicated ratios (dotted traces) at −30 °C. The spectrum for the 1:6 mixture was used to calculate the line width and the principal elements of the g and A tensors of the spin label, which gave the corresponding fit (middle panel, red trace). The spectrum from the 7:0 mixture was fitted theoretically with these spectral parameters for the two C2-symmetry-related nitroxides that are oriented relative to each other as defined by the Euler angles α, β, and γ as described under “Experimental Procedures” (29–31). This resulted in eight sets of symmetry-equivalent Euler angles that fit the data best, all yielding an identical inter-spin distance of 14.3 Å. Only one set of Euler angles is shown here. D, DEER data for membrane-inserted BAK in the absence of spin dilution. DEER experiments were carried out without spin dilution with the indicated membrane-inserted BAK proteins. The DEER data (left column) were analyzed by the DeerAnalysis program using Tikhonov regularization, resulting in distance distribution functions on the right panel (red traces), which could be best fitted with two Gaussian models (blue traces, right panel) with the average inter-spin distance <r> and the standard deviation for the shorter distances indicated. The dotted lines superimposed to the DEER signals are the calculated DEER signal for the distance fit. E, angular clustering of certain residues in helix α5. Side chains of residues Ser-122, Gly-124, Ala-128, Arg-135, Gln-142, and Arg-143, which show spin-spin interactions for the corresponding R1 residues, are clustered on one side of helix α5. Ser-122 is in the loop just upstream of α5 N terminus. F, anti-parallel arrangement of two α5 helices in the BAK homodimer. The C_α carbon atoms of the three residues indicated are shown in spheres on α5 helices along with the inter-spin distances for the symmetry-related spin label pairs in the BAK homology model of BAX BH3-in-groove homodimer model (6).

FIGURE 6.

Proximity of helix α6 residues in membrane-inserted BAK oligomers. A, DEER data for residues 149R1, 151R1, and 154R1 in the absence of spin dilution. DEER experiments were carried out for the indicated proteins without spin dilution. The DEER data (left column) were analyzed by the DeerAnalysis program using Tikhonov regularization, resulting in the distance distribution functions on the right panel (red traces). The black dotted lines superimposed to the DEER signals (red traces, left panel) are the calculated DEER signal for the distance fit. Results by two Gaussian models are superimposed (black traces, right panel) with the average inter-spin distance, the standard deviation and the percent of the spin pairs for the distances indicated. B, proximity of helix α6 residues 162R1 and 163R1 in BAK oligomers. The area-normalized EPR spectra of the spin-diluted samples (3:4 mixture, black trace) of the indicated membrane-inserted BAK are superimposed to those of the undiluted samples (7:0 mixture, red trace). The spectra for 162R1 and 163R1 were acquired at −30 and 22 °C, respectively. The data could be best-fitted with one or two Gaussian distance distributions (right panel). Residue 162R1, with a spin labeling efficiency of 74.2 (±0.6)%, had the interacting spin pairs close to the expected value of 55% (52.3% interacting spins for the single Gaussian fit (red trace) and 50.4% (=10.4 + 40.0) for the double Gaussian fit (black trace). Similarly, residue 163R1 (58.6 (±1.1)% labeling efficiency), resulted in ∼35% interacting spin pairs (34% expected) in the data fitting. C and D, pseudo parallel or pseudo anti-parallel arrangement of two neighboring α6 helices in the BAK oligomer. Two tilted α6 helices are arranged side by side (C) or in a head-to-head orientation (D) with their C termini in close proximity. The average inter-spin distances (dotted arrows) are shown for the indicated pairs of residues (C_α carbon atoms in black spheres).

FIGURE 7.

BAK forms symmetric BH3-in-groove homodimers that associate by α6:α6 interactions to generate higher order oligomers in membrane, juxtaposing the C termini of helices α3 and α5, respectively, between two neighboring homodimers. A, association of the BH3-in-groove homodimers in the membrane is schematically shown. The C_α-carbon atoms of the residue locations chosen for cysteine substitution mutation are shown in colored spheres (red and black for Cys-69 and Cys-111, and green for Cys-96, Cys-143, and Cys-162, respectively). The α-helices and amino acid locations are indicated with primed and unprimed numbers for the two polypeptide chains in two gray shades, respectively. The double headed arrows indicate cross-linkable cysteines. B, spin dilution experiment for 96R1, a C-terminal residue of helix α3. The spin dilution experiment was carried out with membrane-inserted BAK 96R1 samples as indicated. The best fitted one-Gaussian distance distribution (assuming 57.3% interacting and 42.7% non-interacting spins) resulted in the average distance and S.D. indicated. BAK 96R1 had a spin labeling efficiency of 84.9 (±1.0)% and a specific relative percent release activity of 96.0 (±6.1)% (see Fig. 4). C and D, rationale for the detection of various BAK homodimers and homooligomers by disulfide bond formation. The letter M represents a monomer of BAK. The letter C represents cysteine residues substituted at the indicated locations of BAK. D₂ represents dimers of BAK with two disulfide bonds formed between the monomers, resulting in a slightly reduced electrophoretic mobility than other dimers. M_2n represents oligomers of even-numbered BAKs formed by the polymerization of BAK homodimers. This figure was adapted from Dewson et al. (7). E, dye releasing activity of the cysteine substitution BAK mutant proteins. Liposome dye release experiments were carried out with the indicated cysteine-substituted sBAK-ΔC-His proteins (5 nm) in the presence of 25 nm N-terminally His-tagged p7/p15 Bid as described under “Experimental Procedures.” The average values of two experiments are shown with the error ranges. F–I, cross-linking of BAK in membrane-inserted or in solution state. The indicated BAK single, double, and triple cysteine mutants in solution state (G and I) or in the membrane-inserted state (F and H) were cross-linked by copper(II)/phenanthroline complex (Cu(Phe)₃) (34) and were analyzed by Western blotting analysis against BAK after PAGE under a reducing (data not shown) or a non-reducing condition (F–I). In lanes 5 and 6 in F and G, 69C+111C represents a mixture of equal quantity of two single cysteine substitution mutant proteins either in solution (G) or in the membrane-inserted state (F). D₁, D₂, and 3x, 4x, etc., represent BAK single disulfide-bonded dimer, double disulfide-bonded dimer, trimer, tetramer, etc., respectively.

FIGURE 8.

Theoretical models of the BAK α3:α3′, α5:α5′ oligomerization interface on a flat surface and in a pore. A, the best geometry of BAK dimers on a flat membrane surface satisfying the distance constraints for the α3:α3′, α5:α5′ oligomerization interface. Two BAK homodimers, each consisting of two α2-α5 polypeptides in two shades of gray, are brought to each other, satisfying the distance constraints for the 96R1–96R1 and 143R1–143R1 spin pairs at the α3:α3′, α5:α5′ oligomerization interface with the assumption that the R1-R1 distances are the same as the C_α-C_α distances for the pairs. In each homodimer shown in a top view (upper panel) and a side view (bottom panel), the C2-symmetry axes are perpendicular to a horizontal line (line a) on the membrane surface (coplanar with the page). In the best geometry simulated, the angle between line a and the lines that connect the two symmetry-related 143C_α atoms in each homodimer (b lines) was ∼15° with the distance between the two C2-symmetry axes of ∼45 Å. B, a theoretical model of a hexamer of BAK homodimers. Six BAK BH3-in-groove homodimers (i.e. six α2-α5 homodimers in side view of A) are arranged in a hexagonal geometry, forming a pore (only two homodimers are shown). In this geometry, BAK molecules are arranged in a way that the horizontal projections of the b lines of the six homodimers form a hexagon with the edge length of 50 Å, where the indicated distance between the C2-axes would be ∼43 Å (top view). The radius of the aqueous pore formed by the hydrophilic surface (helices α2-α3) of the BAK homodimers will be ∼25 Å due to the thickness of the homodimer (∼18 Å). A vertical section of a putative lipidic pore, which would interact with the hydrophobic surface of the hexameric arrangement of the BAK dimers, is schematically shown (side view, bottom).