BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.

中文翻译：

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膜上促凋亡 BAK 的动态重构

BAK 和 BAX 是内在细胞凋亡的效应子，它们各自都经历了重大的重新配置，形成一个激活的构象异构体，该构象异构体自结合以破坏线粒体并导致细胞死亡。然而，在膜存在下这种重新配置的动态结构机制尚未完全阐明。为了探索膜结合 BAK 的变态，我们采用了氢-氘交换质谱 (HDX-MS)。BAK 在包含线粒体脂质的脂质体上的 HDX-MS 谱与已知的非活性 BAK 溶液结构一致。激活后，HDX-MS 解决了 BAK 中的主要重新配置问题。由我们的 HDX-MS 分析引导的诱变显示 BCL-2 同源性 (BH) 4 结构域保持 BAK 的非活性构象，破坏该结构域足以使 BAK 活化。而且，BAK寡聚化结构域之前的整个N末端区域在激活后变得无序并且在激活的寡聚体中保持无序。去除无序的 N 末端并没有损害，而是稍微增强了 BAK 介导的脂质体和线粒体的膜通透性。总之，我们的 HDX-MS 分析揭示了对膜上 BAK 活化动态性质的新见解，这可能为治疗靶向提供新的机会。