Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095.
Jules Stein Eye Institute, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2018 Jan 9;115(2):E172-E179. doi: 10.1073/pnas.1715464115. Epub 2017 Dec 26.
The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pK of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.
电压依赖性阴离子通道（VDAC）是线粒体外膜中最丰富的蛋白质，构成了细胞质和线粒体之间离子和代谢物交换的主要途径。越来越多的证据支持 VDAC 在线粒体代谢调节和细胞凋亡中的作用，其中 VDAC 寡聚化与这些过程有关。在此，我们通过双电子电子共振和天然质谱法报告了一种由鼠源 VDAC1（mVDAC1）组成的特定 pH 依赖性二聚体。使用四个单自旋标记的 mVDAC1 突变体的分子间距离来生成低 pH 二聚体的模型，确定了界面处存在残基 E73。这种二聚体排列与以前描述的任何寡聚状态都不同，并且它以 pH 值为陡峭函数形成，表观 pK 值为 7.4。此外，使用天然 MS 确定了单体-二聚体平衡亲和力常数，在低 pH 值下二聚体亲和力增强近 8 倍。将 E73 突变为丙氨酸或谷氨酰胺会严重降低寡聚化，证明质子化 E73 在增强二聚体形成中的作用。基于这些结果以及 E73 在 VDAC 生理学中的已知重要性，VDAC 二聚体可能在细胞应激时细胞质酸化导致的线粒体代谢调节和细胞凋亡中发挥重要作用。
