Permeabilization of the outer mitochondrial membrane by pore-forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro-apoptotic Bak during pore formation, high-resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX-MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high-resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3-only proteins. Furthermore, we determined the first high-resolution structure of the Bak transmembrane helix. Upon activation, α-helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane-bound state. In line with this finding, comparative protein folding experiments with Bak and anti-apoptotic BclxL suggest that α-helix 1 in Bak is a metastable structural element contributing to its pro-apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α-helix 1 yielded Bak variants with delayed pore-forming activity. These insights will contribute to a better mechanistic understanding of Bak-mediated membrane permeabilization.

中文翻译：

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促凋亡 Bak 在脂膜上构象转变的高分辨率分析

成孔 Bcl2 蛋白对线粒体外膜的透化是诱导细胞凋亡的关键步骤。尽管大量数据表明促凋亡 Bak 在孔形成过程中发生了整体构象变化，但膜环境中的高分辨率结构细节仍然稀疏。在这里，我们在脂质纳米盘中使用 NMR 和 HDX-MS（氢氘交换质谱）来获得重要的高分辨率结构见解，了解膜上 Bak 的构象变化，这些变化依赖于仅 BH3 蛋白的直接激活。此外，我们确定了 Bak 跨膜螺旋的第一个高分辨率结构。激活后，Bak 可溶性结构域中的 α-螺旋 1 从蛋白质上解离，并采用展开的动态潜在膜结合状态。与这一发现一致，Bak 和抗凋亡 BclxL 的比较蛋白质折叠实验表明，Bak 中的 α-螺旋 1 是一种亚稳态结构元件，有助于其促凋亡特征。因此，旨在稳定 α-螺旋 1 的诱变实验产生了具有延迟成孔活性的 Bak 变体。这些见解将有助于更好地理解 Bak 介导的膜透化作用的机制。