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. 2013 Mar;9(3):163-8.
doi: 10.1038/nchembio.1166. Epub 2013 Jan 20.

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Ariele Viacava Follis  1 , Jerry E Chipuk, John C Fisher, Mi-Kyung Yun, Christy R Grace, Amanda Nourse, Katherine Baran, Li Ou, Lie Min, Stephen W White, Douglas R Green, Richard W Kriwacki
Affiliations

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Ariele Viacava Follis et al. Nat Chem Biol. 2013 Mar.

Abstract

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the antiapoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique among BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules BAX and BAK. Structural investigations using NMR spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the antiapoptotic BCL-2 repertoire to sensitize for death receptor-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

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Figures

Figure 1
Figure 1
Structural and dynamic characterization of the BCL-xLΔLΔC·PUMABH3 complex in solution. a. Scheme illustrating the mechanism by which p53 regulates apoptosis through interactions with DNA in the nucleus and BCL-2 family proteins in the cytosol. Increasing numerals denote the sequence of events involved in this process. b. Sequence alignment of BH3 domains, color-coded according to conservation (bold green: conserved; green: highly conserved; olive: partially conserved; red: unique). The consensus motif is indicated: ϕ hydrophobic residue; g Gly, Ser or Ala; L Leu; r usually Arg or Lys; Φ hydrophobic residue; D Asp; and e usually Glu or Asp. Unique residues in the PUMA BH3 domain are Gln70 and Trp71; residue numbers for PUMA are given at the top. These sequences correspond to the synthetic peptides employed in this study. c. Solution structure of the BCL-xLΔLΔC·PUMABH3 complex; ribbon representation of the lowest-energy structure (left) and alignment of the 20 lowest-energy structures (right). BCL-xLΔLΔC is colored blue and PUMABH3 is colored red. d. Structural representation of 1H-15N NMR chemical shift perturbations caused by PUMABH3 binding to BCL-xLΔLΔC. e. Equivalent representations for the BCL-xLΔLΔC·BADBH3 complex (PDB: 1G5J; BMRB entry: 6578). f. Sequence dependence of random coil index order parameter (RCI S2) for free BCL-xLΔLΔC (blue), BCL-xLΔLΔC·BADBH3 (light blue), and BCL-xLΔLΔC·PUMABH3 (red). The BH3 interaction site is highlighted above the graph (dark gray) within a schematic representation of the protein’s α-helices. g. Sequence dependence of {1H}-15N HetNOE values for the same proteins as illustrated in f. Error bars are inversely proportional to the signal-to-noise ratio of each resonance.
Figure 2
Figure 2
Mechanism of PUMA binding-induced p53 release from BCL-xL. a. Crystal structure of BCL-xLΔC domain-swapped dimer bound to PUMABH3. The two subunits of BCL-xLΔC are colored dark blue and light blue, respectively, and the eight α-helices of one globular core of the dimer (right side) are labeled α1-α5 and α6′-α8′. The two molecules of PUMABH3 are colored red (front, right) and light red (back, left), respectively. The imidazole ring of His113 of BCL-xL and the indole ring of Trp71 of PUMABH3 that are engaged in a π-stacking interaction are indicated. b. Fluorescence polarization analysis of titrations of p53SM 1-360 into fluorescently labeled BCL-xLΔC (F-BCL-xLΔC), isolated or previously bound to a slight molar excess of different BH3 peptides from PUMA, BIM, BID, BAD, BAX, BAK or HRK as indicated. Error bars represent the standard error of the mean of five independent titrations. c. Sequence dependence of {1H}-15N HetNOE values for BCL-xLΔLΔC in complex with PUMABH3 W→A (orange) and BCL-xLΔLΔCH→A in complex with wild-type PUMABH3 (blue; the mutation site is marked with an asterisk). The values observed for BCL-xLΔLΔC·PUMABH3 (Fig. 1g) are illustrated with a dashed red line. d–f. Overlaid 2D 1H-15N TROSY spectra of 100 μM 15N-BCL-xLΔLΔC bound to unlabeled PUMABH3 (d), PUMABH3 W→A (e) or 15N-BCL-xLΔLΔCH→A bound to PUMABH3 (f) in the absence (blue) and presence (red) of a 1.5 molar excess of p53SM 1-360. g. Surface representation of apo BCL-xL highlighting the non-overlapping nature of its surfaces that bind to BH3 domains, including PUMABH3 (green) and p53 (orange).
Figure 3
Figure 3
PUMA-induced p53 release from BCL-xL differentially regulates apoptotic pathways. a. His-tagged BCL-xLΔC·p53UVIP complexes were combined with PUMABH3 or the indicated direct activator BH3 domain peptides (10, 50 and 100 nM concentrations); His-BCL-xLΔC was then isolated using nickel affinity beads. His-BCL-xLΔC and associated p53 were detected after SDS-PAGE by western blot analyses. b. His-BCL-xLΔC·p53UVIP complexes (20 nM) were combined with bak−/−bax−/− mitochondria in the presence of BAX and indicated derepressor BH3 domain peptides or PUMA (40 nM), followed by fractionation and either nickel affinity pull-down of His-BCL-xLΔC, SDS-PAGE and western blot analyses for p53 and BCL-xL (upper panels), or western blot analysis for mitochondrial (labeled “p”) or released (labeled “s”) cytochrome c (lower panels). c. BCL-xLΔC·p53UVIP complexes (the two components were mixed at 100 nM and 10 nM concentrations respectively) were combined with bak−/−bax−/− liver mitochondria in the presence of BAX (20 nM) and the indicated derepressor BH3 domain peptides (1 μM) or PUMA (100 nM) before fractionation, SDS-PAGE and western blot analyses for cytochrome c.
Figure 4
Figure 4
Trp71 (W71) of PUMA is required for p53-dependent, DNA damage-induced apoptosis. a. puma−/− MEFs were transiently transfected with pCMVneoBam, pCMV5neoBam-FLAG-PUMA or pCMV5neoBam-FLAG-PUMA W71A (PUMAW→A), allowed to recover for 24 h, treated with TNF (0, 5 and 10 ng/ml) and cycloheximide (10 μg/ml) for 6 hours and analyzed by AnnexinV-PE staining and flow cytometry for apoptosis. US9-GFP was cotransfected and only GFP positive cells were analyzed. b. puma−/− MEFs were transiently transfected with pCMVneoBam, pCMVneoBam-FLAG-PUMA or pCMVneoBam-FLAG-PUMA W71A (PUMAW→A), recovered for 24 h, treated with UV irradiation (0, 2.5 and 5 mJ/cm2) and analyzed for apoptosis as above. c. Lysates from (b) were subjected to co-immunoprecipitation with anti-FLAG and analyzed by SDS-PAGE and western blot for FLAG-PUMA (wild type or W71A), BCL-xL and p53. Error bars in a and b represent the standard deviation calculated from at least three independent experiments. d. Schematic illustration of the mechanism by which PUMA induces unfolding within α2 and α3 of BCL-xL, which is associated with p53 release. The formation of a π-stacking interaction between His113 of BCL-xL (blue pentagon shapes) and Trp71 of PUMA (magenta geometric shapes) is associated with unfolding of α2 and α3 (α3* in the upper right). BCL-xL is represented as a multi-color hexagon, with the edges representing its α-helices, as marked, p53 as a yellow oval and PUMA in magenta in unbound form as a wavy line and as a cylinder when bound to BCL-xL.
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