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Deciphering molecular specificity in MCL-1/BAK interaction and its implications for designing potent MCL-1 inhibitors

Cell Death & Differentiation volume 32, pages 991–999 (2025)Cite this article

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Abstract

The intricate interplay among BCL-2 family proteins governs mitochondrial apoptosis, with the anti-apoptotic protein MCL-1 primarily exerting its function by sequestering the pore-forming effector BAK. Understanding the MCL-1/BAK complex is pivotal for the sensitivity of cancer cells to BH3 mimetics, yet the precise molecular mechanism underlying their interaction remains elusive. Herein, we demonstrate that a canonical BH3 peptide from BAK inadequately binds to MCL-1 proteins, whereas an extended BAK-BH3 peptide with five C-terminal residues exhibits a remarkable 65-fold increase in affinity. By elucidating the complex structures of MCL-1 bound to these two BAK-BH3 peptides at 2.08 Å and 1.98 Å resolutions, we uncover their distinct binding specificities. Notably, MCL-1 engages in critical hydrophobic interactions with the extended BAK-BH3 peptide, particularly at an additional p5 sub-pocket, featuring a π-π stacking interaction between MCL-1 Phe319 and BAK Tyr89. Mutations within this p5 sub-pocket substantially disrupt the MCL-1/BAK protein-protein interaction. Furthermore, the p5 sub-pocket of MCL-1 significantly influences the efficacy of MCL-1 inhibitors. Overall, our findings elucidate the molecular specificity underlying MCL-1 binding to BAK and underscore the significance of the p5 hydrophobic sub-pocket in their high-affinity interaction, thus providing novel insights for the development of BH3 mimetics targeting the MCL-1/BAK interaction as potential therapeutics for cancer treatment.

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Fig. 1: Binding affinity of BAK-BH3 peptides to anti-apoptotic BCL-2 proteins.
Fig. 2: Structures of MCL-1 with BAK-BH3long and BAK-BH3short.
Fig. 3: Comparison of the interactions of BAK-BH3 with MCL-1 and BCL-XL.
Fig. 4: Effect of MCL-1 and BAK mutations on the MCL-1/BAK interaction.
Fig. 5: Effect of the MCL-1 mutation F319A on the MCL-1 inhibitor A-1210477.

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Data availability

The coordinates and structure factors are deposited in the Protein Data Bank under the accession codes 8Y1Y (MCL-1/BAK-BH3long structure), 8Y1Z (MCL-1/BAK-BH3short structure) and 8Y20 (MCL-1/A-1210477 structure). Other data generated in this study are provided in the article and in the Supplementary Materials.

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Acknowledgements

We thank the staff at BL17B1/BL18U1/BL19U1 beamlines at SSRF of the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, for providing technical support in X-ray diffraction data collection and analysis. We thank Department of Clinical Pharmacology, Xiangya Hospital, Central South University for providing Biacore 8 K instrument and technical support.

Funding

The present study was financially supported by the Natural Science Foundation of China (82470176, 82273496, 31900880 and 82172654), Hunan Provincial Science and Technology Department (2021RC4012), Changjiang Scholars Award Program of Ministry of Education, Natural Science Foundation of Hunan Province (2023JJ20092 and 2023JJ30863) and Central South University Innovation-Driven Research Program (2023CXQD076).

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Author notes

  1. These authors contributed equally: Hudie Wei, Haolan Wang, Shuang Xiang.

Authors and Affiliations

  1. Department of Oncology, NHC Key Laboratory of Cancer Proteomics & State Local Joint Engineering Laboratory for Anticancer Drugs, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China

    Hudie Wei, Haolan Wang, Jiaqi Wang, Lingzhi Qu, Xiaojuan Chen, Ming Guo & Yongheng Chen

  2. State Key Laboratory of Bioactive Molecules and Druggability Assessment, International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Discovery of Chinese Ministry of Education, Guangzhou City Key Laboratory of Precision Chemical Drug Development, School of Pharmacy, Jinan University, Guangzhou, China

    Shuang Xiang & Xiaoyun Lu

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Contributions

YC and Wei H conceived the project. MG, Wang H, SX, LQ, JW, and Wei H performed experiments. M.G. and Wang H performed data collection and structure determination. XC and XL helped with data analysis. MG, XL, YC, and Wei H prepared the paper.

Corresponding authors

Correspondence to Ming Guo, Xiaoyun Lu or Yongheng Chen.

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The authors declare no competing interests.

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This study was approved by the ethics committee of the affiliated Xiangya Hospital of Central South University. All methods were performed in accordance with the relevant guidelines and regulations. Human cell lines were treated as described in the methods section of this manuscript. This study did not involve human subjects research or experiments on live vertebrates.

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Wei, H., Wang, H., Xiang, S. et al. Deciphering molecular specificity in MCL-1/BAK interaction and its implications for designing potent MCL-1 inhibitors. Cell Death Differ 32, 991–999 (2025). https://doi.org/10.1038/s41418-025-01454-2

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