Abstract
Under conditions of genotoxic stress, human p53 activates the apoptotic effectors BAX or BAK to result in mitochondrial outer-membrane permeabilization and apoptosis. Antiapoptotic BCL-2 family member BCL-xL opposes this activity by sequestering cytosolic p53 via association with its DNA-binding domain, an interaction enhanced by p53 tetramerization. Here we characterized the BCL-xL–p53 complex by NMR spectroscopy and modulated it through mutagenesis to determine the relative contributions of BCL-xL's interactions with p53 or other BCL-2 family proteins to the BCL-xL–dependent inhibition of UV irradiation–induced apoptosis. Under our experimental conditions, one-third of the antiapoptotic activity of BCL-xL was mediated by p53 sequestration and the remaining two-thirds through sequestration of proapoptotic BCL-2 family members. Our studies define the contributions of cytosolic p53 to UV irradiation–induced apoptosis and provide opportunities to explore its contributions to other p53-dependent apoptotic signaling pathways.
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Acknowledgements
The authors acknowledge C.R. Grace (St. Jude Children's Research Hospital) for assistance with NMR experiments, C.-G. Park (St. Jude Children's Research Hospital) for assistance with protein preparation and isotope labeling and J. Chipuk (Mount Sinai Medical Center) for helpful discussion and advice. This work was supported by US National Institutes of Health (NIH) grants R01CA082491 and 1R01GM083159 (to R.W.K.), NIH R01GM52735 and R01GM96208 (to D.R.G.), US National Cancer Institute Cancer Center Support grant P30CA21765 (at St. Jude Children's Research Hospital) and the American Lebanese Syrian Associated Charities. A.V.F. is supported as the recipient of the Neoma Boadway Fellowship from St. Jude Children's Research Hospital.
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A.V.F. and L.O. contributed to NMR experiments and data analysis; A.V.F. contributed to structural calculations and biophysical and biochemical assays; A.V.F., F.L. and K.B. contributed to cellular assays; A.V.F., D.R.G. and R.W.K. wrote the manuscript. All authors contributed to different aspects of the design and conception of this study.
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Integrated supplementary information
Supplementary Figure 1 NMR titrations between [15N]BCL-xLΔC and unlabeled p53 domains.
Overlaid full views of 1H-15N-TROSY spectra of 15N-BCL-xLΔC upon titration of unlabeled p53 NTD (a); p53 DBD (b); p53 N-D (c); p53 N-D-T (d); p53 TET (e). For panels a trough d the displayed spectra follow a blue to red transition color scheme at increasing p53 molar ratios. 15N-BCL-xLΔC concentration was 100 μM in all titrations (80 μM in e); p53 NTD (a) and p53 DBD (b) concentrations were 0, 10, 20, 40, 75, 110, 150, 200, 250, 300 μM; p53 N-D (c) concentrations were 0, 10, 25, 50, 75, 120, 180, 250, 300 μM; p53 N-D-T (d) concentrations were 0, 10, 25, 50, 75, 110, 150, 200 μM. f. Sequence plot of BCL-xLΔC resonance intensities relative to its free state in the presence of 180 μM p53 N-D (green trace) or 200 μM p53 N-D-T (purple trace).
Supplementary Figure 2 1H-15N TROSY titrations of unlabeled BCL-xLΔC into various 15N-labeled p53 constructs.
Titration of p53 NTD (a); p53 DBD (b) p53 N-D (c) p53 N-D-T (d). The displayed spectra follow a blue to red transition color scheme at increasing BCL-xLΔC molar ratios. The concentration of p53 NTD, p53 DBD and p53 N-D was 100 μM; that of p53 N-D-T 180 μM. BCL-xLΔC was titrated into 15N-p53 NTD (a) at 0, 50, 100, 200 μM; into 15N-p53 DBD (b) at 0, 25, 50, 100, 200, 300 μM; into p53 N-D (c) at 0, 25, 50, 100, 150, 200 μM; into p53 N-D-T (d) at 0 and 360 μM. e. Sequence plot of chemical shift perturbations detected in p53 N-D in the presence of 200 mM BCL-xLΔC. f. Mapping of the data displayed in panel e onto the p53 DBD structure indicating residues that showed backbone amide chemical shift perturbation > 0.06 ppm.
Supplementary Figure 3 Measurements of paramagnetic relaxation enhancements (PREs) for BCL-xLΔC–p53 DBD structure determination.
a. Overlaid 1H-15N-TROSY spectra of 15N-p53 DBD (100 μM) in the presence of reduced (blue) or oxidized (red) Cys151-MTSL labeled BCL-xLΔC (150 μM) and (b) of 15N-BCL-xLΔC (100 μM) in the presence of Zn2+ (blue) or Co2+-coordinating (red) p53 DBD (150 μM). c. Mapping onto the structure of p53 DBD of the paramagnetic relaxation enhancements (PREs) observed in a. d. Equivalent mapping of PREs observed in b onto the structure of BCL-xL.
Supplementary Figure 4 Intermolecular NOEs observed between [2H, 15N]p53 DBD and BCL-xLΔC at long NOE mixing time (160 ms).
The top two panels display slices of the 1H-15N-TROSY-NOESY 3D spectrum for selected resonances of 2H/15N-p53 DBD (450 μM) in the absence (a) and presence (b) of BCL-xLΔC at a 2:1 molar ratio. The bottom panel (c) displays slices of the 1H-15N-TROSY-TOCSY spectrum of free 15N-BCL-xL for the residues to which the observed inter-molecular NOEs were assigned. The inter-molecular NOESY peaks in the 3D NOESY spectrum were assigned by inference, with reference to the 15N-TROSY-TOCSY spectrum of free 15N-BCL-xL and with knowledge of preliminary structures based upon extensive PRE data. In a few cases, there are p53 binding-induced chemical shift changes between the two spectra (marked with an asterisk). d. Structure mapping of the p53 DBD (top) and BCL-xL (bottom) residues that exhibited intermolecular NOEs shown as spheres and color coded based on their reciprocal assignment.
Supplementary Figure 5 Conformational rearrangements take place within BCL-xL upon complex formation with p53 DBD.
a. Bundle of 20 lowest energy structures of apo BCL-xLΔC calculated with restraints obtained from 2H/13C/15N-BCL-xLΔC with selective 1H-labeling of Isoleucine, Leucine, Valine methyl moieties. b.. Equivalent representation of the BCL-xLΔC structure obtained from an identically labeled sample in the presence of p53 DBD. c. Overlay between the lowest energy structures in panels a (cyan) and b (pink). Flexible regions are omitted for clarity. A marked rearrangement of the α2-3 region of BCL-xL is observed upon binding of p53 DBD (highlighted with a red ellipse). d,e. Slices corresponding to the C-terminus of α2 of from the 1H-15N-TROSY-NOESY 3D spectrum of 2H/13C/15N-BCL-xLΔC with selective 1H-labeling of Isoleucine, Leucine, Valine methyl moieties in its apo form (d) and in the presence of p53 DBD (e) highlighting the subtle changes in local stabilization of α-helical structure that takes place in this region of BCL-xL upon binding of p53 DBD. Patterns of NOE cross-peaks indicative of a-helical structure are highlighted by dashed lines (red: i - i±1; yellow: longer range) and the approximate limits of strong a-helical NOE patterns are marked at the top of each panel along with residue labels. All samples contained each protein component at 600 μM. f. Sequence mapping of chemical shift-derived backbone dihedral restraints (calculated with the program Talos)58 for apo BCL-xLΔC (blue) and BCL-xLΔC bound to p53 DBD (red). The position of individual a-helices is marked above the plot. An increase in a-helical dihedral angles is observed at the C-terminus of α2 when BCL-xL is bound to p53-DBD.
Supplementary Figure 6 Structure-based mutagenesis of interfacial residues within the p53 DBD–BCL-xL complex.
a. Binding titrations between fluorescently labeled, wild-type BCL-xLΔC (F-BCL-xLΔC) and wild-type or mutated p53 N-D-T. Error bars, s.e.m. (n = 3 titrations). b. Mapping of the studied mutations on the p53 DBD surface depicted in the same orientation as Figure 4c. c. Competition experiments between F-BCL-xLΔC and unlabeled wild-type or mutated BCL-xLΔC for p53 binding. Error bars, s.e.m. (n = 3 titrations). d. Mapping of the studied mutations on the BCL-xL surface depicted in the same orientation as Figure 4d. e. Fluorescence polarization titration between F-BCL-xLΔC, pre-incubated with 1mM ABT-737 and p53 N-D-T. Error bars, s.e.m. (n = 3 titrations). f. Fluorescent polarization titration between F-BCL-xLΔC and full-length, wild-type human p53 expressed from Sf9 insect cells using a Baculovirus expression system (Bac-p53). Error bars, s.e.m. (n = 3 titrations). The measured binding affinity between these two proteins is equivalent to that between F-BCL-xLΔC and the p53 N-D-T bearing core stabilizing mutations.
Supplementary Figure 7 Binding titrations between fluorescently labeled, double-stranded oligonucleotides and wild-type or mutant p53 N-D-T.
a. Binding mode between p53 DBD (light grey) and DNA. The minimal binding unit is composed of one double stranded consensus sequence and two p53 DBD. Promoter regions in p53 target genes contain two closely spaced consensus sequences and are bound by p53 tetramers (in a 'dimer of dimers' fashion). b. Sequences of the studied oligonucleotides. The partial consensus segments are highlighted in bold characters in each sequence and nucleotides that differ between the two promoters are highlighted in red. The structural depictions at the bottom illustrate how sequence differences between the promoter regions correlate with residues within p53 whose mutation differentially altered the binding affinity for the two promoters. c. Fluorescence polarization binding titrations between wild-type or mutant p53 N-D-T and fluorescent p21 promoter. d. Analogous representation of binding titrations to the puma promoter. e. Structure representation of p53 bound to DNA highlighting interface regions and mutated residues that overlap with the p53 – BCL-xL binding interface. f. Competition experiments between F-BCL-xLΔC and an unlabeled p21 promoter double stranded oligonucleotide for p53 binding g. Fluorescence polarization titrations between Bac-p53 and fluorescently labeled DNA. Bac-p53 or p53 N-D-T display comparable binding affinities in vitro to these two oligonucleotides. Data in panels c,d,f,g represent the average of three independent titrations, error bars indicate s.e.m.
Supplementary Figure 8 Functional relevance of p53 mutations
a. Correlation between p21 promoter binding free energies and p21 transcription levels in yeast, as assessed by Kato et al.39, by wild-type and mutant p53. b. Equivalent analysis for the average DNA binding free energies (p21, puma promoter) and the average transcription levels in yeast of all eight p53 target genes analyzed by Kato et al.39. c. Western blot analysis of cytosolic (C), nuclear (N) and heavy membrane (HM) H1299 cell fractions transfected with empty vector or the indicated p53 constructs. Membranes were probed for p53, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, cytosolic marker) and Lamin B (nuclear marker). d. Effect of p53 mutations on its ability to activate BAX in vitro in a large unilamellar vesicle (LUV) permeabilization assay, analogous to the one reported in Figure 7a d but in the absence of BCL-xL inhibiting BAX activation. All tested mutants induced lower BAX activation compared to wild-type p53, indicating a possible overlap of interaction interfaces between BCL-xL or BAX and p53. Reduction in BAX activation was particularly pronounced for the H178D E180K double mutant, and roughly equivalent to the additive detrimental effect of each individual mutation. e. LUV permeabilization assay testing the ability of wild-type or mutant BCL-xLΔC to prevent liposome permeabilization by active BAX. Unlike the one displayed in Figure 7a, in this assay no agent was introduced to block the BH3 binding groove of BCL-xL (BAD BH3, ABT-737) therefore only limited LUV permeabilization was observed in the presence of wild-type or mutant BCL-xLΔC, likely because all these BCl-xL constructs are capable of sequestering partially active BAX by binding its exposed BH3 domain. f. LUV permeabilization assay testing the ability of wild-type or mutant BCL-xLΔC to prevent BAX activation by BIM or BID BH3 constructs. In this experiment, BIM-BID BH3 peptides or BIM-BID BH3-BCL-xL complexes were added to LUV samples at a lower concentration than the total amount of BAX present, therefore minimizing any potential effect of BCL-xL sequestration of active BAX. Both tested BCL-xL mutants showed equal ability to the wild-type protein to inhibit BIM-BID-dependent BAX activation.
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Follis, A., Llambi, F., Ou, L. et al. The DNA-binding domain mediates both nuclear and cytosolic functions of p53. Nat Struct Mol Biol 21, 535–543 (2014). https://doi.org/10.1038/nsmb.2829
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DOI: https://doi.org/10.1038/nsmb.2829
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