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A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C

Cell Death & Differentiation volume 30, pages 794–808 (2023)Cite this article

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Abstract

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4–α5, α5–α6, or α6–α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

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Fig. 1: Activation-associated binding of BAKΔTM to the MOM requires movement of multiple helices.
Fig. 2: BAK helical fragments other than α9 target mitochondria.
Fig. 3: The first or second most populated conformations of tandem helix peptides and selected variants.
Fig. 4: BAK tandem helices directly permeabilize the MOM and induce cell death.
Fig. 5: Preferential binding of BAK peptides to MOM lipids is essential for MOMP.
Fig. 6: BAK peptide alterations modulate lipid binding and cytochrome c release.
Fig. 7: Effect of α9 or proline insertion on BAK tandem peptide-induced membrane permeabilization and cytotoxicity.
Fig. 8: Effect of helix shortening on MOMP and cell killing.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files.

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Acknowledgements

We thank David Toft, Richard Youle, Qian Liu, and Kalle Gehring for gifts of reagents; Eric Roush (Cytiva) for advice regarding surface plasmon resonance analysis; Gregory Gores, Roderick Brown, and Husheng Ding for helpful discussions; and the two anonymous reviewers for insightful suggestions. We also acknowledge the computing resources provided by the University of Minnesota Supercomputing Institute and the Mayo Clinic high-performance computing facility at the University of Illinois Urbana-Champaign National Center for Supercomputing Applications.

Funding

This work was supported in part by grants from the National Cancer Institute (R01 CA166741, R01 CA225996 and P30 CA015083).

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Author notes

  1. These authors contributed equally: Haiming Dai, Scott H. Kaufmann.

Authors and Affiliations

  1. Division of Oncology Research, Mayo Clinic, Rochester, MN, 55905, USA

    Haiming Dai, Kevin L. Peterson, Karen S. Flatten, X. Wei Meng, Annapoorna Venkatachalam, Cristina Correia & Scott H. Kaufmann

  2. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, 55905, USA

    Haiming Dai, X. Wei Meng, Cristina Correia, Yuan-Ping Pang & Scott H. Kaufmann

  3. Anhui Province Key Laboratory of Medical Physics and Technology, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China

    Haiming Dai

  4. Hefei Cancer Hospital, Chinese Academy of Sciences, Hefei, 230031, China

    Haiming Dai

  5. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA

    Marina Ramirez-Alvarado

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Contributions

Designed study: HD and SHK Conducted experiments: HD, KLP, KSF, XWM, AV, Y-PP. Analyzed data: HD, CC, MR-A, Y-PP, SHK. Wrote manuscript: HD, SHK. Edited and approved manuscript: HD, KLP, KSF, XWM, KLP, AV, CC, MR-A, Y-PP, SHK.

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Correspondence to Haiming Dai or Scott H. Kaufmann.

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Dai, H., Peterson, K.L., Flatten, K.S. et al. A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C. Cell Death Differ 30, 794–808 (2023). https://doi.org/10.1038/s41418-022-01083-z

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