Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA.
Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, MO 63110, USA.
Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Oct;1864(10):1269-1279. doi: 10.1016/j.bbalip.2019.06.004. Epub 2019 Jun 5.
Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.
电压依赖性阴离子通道 1（VDAC1）是一种线粒体孔道蛋白，与细胞代谢和细胞凋亡有关，并受许多小分子调节，包括脂质。VDAC1 与固醇结合，包括胆固醇和神经甾体如孕烷醇酮。生化和计算研究表明 VDAC1 结合多个胆固醇分子，但光标记研究仅在 E73 处鉴定出一个胆固醇和神经甾体结合位点。为了确定 VDAC1 中所有神经甾体的结合位点，我们应用两种基于固醇的光标记试剂进行光亲和标记，这两种试剂具有互补的光化学性质：含有脂族重氮的 5α-6-AziP 和含有三氟甲基-苯重氮（TPD）基团的 KK200。5α-6-AziP 和 KK200 标记 E73 口袋内的多个残基，证实了该位点的存在，并绘制了该口袋内固醇的取向。此外，KK200 还标记了另外四个位点，与 VDAC1 与五个胆固醇分子共纯化的发现一致。孕烷醇酮和胆固醇都竞争性地阻止 E73 和另外三个位点的光标记，表明这些是神经甾体和胆固醇共同的常见固醇结合位点。在功能重要的残基 E73 上的结合表明固醇可能在调节 VDAC1 信号和与伴侣蛋白相互作用中起作用。
