Abstract
Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55–C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55–C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.
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Data availability
HXMS data have been deposited to the PRIDE database with identifier code PXD016059. All data generated or analyzed for this study are included in this manuscript and its supplementary files. Source data are available with the paper online.
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Acknowledgements
We thank E. Smith for assistance with figure preparation. The study was funded by NIH grant R35CA197583 and a Leukemia and Lymphoma Society Translational Research Program grant to L.D.W., an American Cancer Society Postdoctoral Fellowship Award to K.J.K. and NIH grant R50CA211399 to G.H.B. Additional support was provided by a research collaboration between J.R.E. and the Waters Corporation. We also thank the Wolpoff Family Foundation, Jim and Lisa LaTorre, the family of Ivo Coll and the Todd J. Schwartz Memorial Fund for their financial contributions to our cancer chemical biology research.
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K.J.K. and L.D.W. designed the study. K.J.K. produced the BFL-1 proteins and performed all biochemical, mitochondrial and cellular experiments. G.H.B. generated peptides. K.J.K. and T.E.W. performed the HXMS analyses under the supervision of J.R.E. L.D.W. and K.J.K. wrote the manuscript, which was reviewed by all co-authors.
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L.D.W. is a scientific co-founder and shareholder in Aileron Therapeutics.
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Extended data
Extended Data Fig. 1 Purification of recombinant full-length BFL-1 constructs.
(a–d) The indicated full-length, N-His6 BFL-1 constructs bearing a C-terminal chitin binding domain (CBD) were expressed in BL21 DE3 cells, purified by chitin affinity resin, subjected to overnight cleavage with hydroxylamine (100 mM), and further purified by SEC. Full-length BFL-1 constructs bearing C55 and C175 (a, c) were isolated as a doublet under non-reducing conditions and as a singlet under reducing conditions, as demonstrated by gel electrophoresis and Coomassie stain. The shaded peak on the FPLC profile indicates the fraction containing pure, full-length BFL-1 protein.
Extended Data Fig. 2 Hydrogen-deuterium exchange profiles of BFL-1ΔC C55 and BFL-1 C55/C175.
(a) Domain map of full-length BFL-1 highlighting the amino acid sequences that correspond to the individual α-helices, the truncation site for BFL-1ΔC C55, and the locations of C55 and C175 (colored in orange). (b-d) The deuterium uptake profiles of BFL-1ΔC C55 (b), BFL-1 C55/C175 without BME (c), and BFL-1 C55/C175 with BME (d) were measured at 10 seconds and 10 minutes of deuterium labeling. The relative deuterium uptake plots demonstrate significant time-dependent exchange in the region spanning the α1-α2 loop to the proximal portion of α4 helix (aa 18-66) and in the C-terminal region from the distal portion of α8 through the C-terminus (aa 144-175). The α5/α6 hydrophobic core of the protein showed relatively less deuterium exchange, and the N-terminal region containing α1 demonstrated little to no deuterium uptake. Data are representative of three independent experiments for BFL-1ΔC C55 and BFL-1 C55/C175 and two independent experiments for BME-reduced BFL-1 C55/C175 (see Supplementary Table 1). All HXMS data used to create this figure can be found in Supplementary Data File 2.
Extended Data Fig. 3 Cellular response to H2O2 treatment.
Cell viability of 293 T cells treated with the indicated concentrations of H2O2 and measured at 24 h. Data are mean ± s.d. of four technical replicates. The experiment was repeated twice using independent cell cultures and treatments with similar results. The sub-cytotoxic dose of 100 μM (asterisk) was chosen for pull-down experiments (Fig. 5d, e). Data for the cell viability plot is available online.
Supplementary information
Supplementary Information
Supplementary Table 1.
Supplementary Data 1
Alignment of BFL-1 amino acid sequences used for phylogenetic analysis.
Supplementary Data 2
HXMS data for analyses of BFL-1 proteins.
Supplementary Data 3
Data for DSF and FP plots and calculations.
Source data
Source Data Fig. 1
Uncropped anti-His6 western blot.
Source Data Fig. 4
Data for liposomal release assay plots.
Source Data Fig. 5
Uncropped anti-His6 and anti-GFP western blots.
Source Data Fig. 5
Data for mitochondrial cytochrome c release and cellular BH3-in-groove assay plots.
Source Data Extended Data Fig. 3
Data for cell viability plot.
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Korshavn, K.J., Wales, T.E., Bird, G.H. et al. A redox switch regulates the structure and function of anti-apoptotic BFL-1. Nat Struct Mol Biol 27, 781–789 (2020). https://doi.org/10.1038/s41594-020-0458-9
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DOI: https://doi.org/10.1038/s41594-020-0458-9
