NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL
NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL
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DOI：
10.1021/bi061577h
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发表时间：
2007-01-16
期刊：
影响因子：
2.9
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Malia, Thomas J.;Wagner, Gerhard
BCL-2家族蛋白是细胞死亡的重要调节因子，在线粒体外膜发挥主要的促凋亡或抗凋亡作用。此前，促凋亡和抗凋亡的Bcl2蛋白已被证明与线粒体外膜的电压依赖性阴离子通道(VDAC)相互作用。VDAC是一种283个残基的完整膜蛋白，它在线粒体外膜上形成一个水孔，代谢物和其他小分子通过这个孔在细胞质和膜间隙之间传递。VDAC在代谢物运输中的重要生命维持功能被认为是由Bcl2家族的蛋白调节的。抗细胞凋亡的Bclx(L)的保护作用可能是通过其与VDAC的相互作用实现的。在这里，VDAC已经被表达、纯化并折叠成适合于核磁共振研究的功能形式。各种生物物理实验表明，胶束结合型VDAC在单体和三聚体之间处于中间交换状态。通过核磁共振光谱、凝胶过滤和化学交联，我们获得了在洗涤剂胶束体系中Bclx(L)与VDAC结合的直接证据。用化学位移微扰和转移交叉饱和的核磁共振方法表征了Bclx(L)的VDAC相互作用区。相互作用区域被映射到推测的螺旋发夹基序的Bclx(L)，它被发现插入到洗涤剂胶束中。我们的结果表明，Bclx(L)可以与一个或两个VDAC分子结合，形成异二聚体和异三聚体。我们对VDAC/Bclx(L)复合体的表征为我们提供了初步的结构洞察，揭示了抗凋亡的Bclx(L)在调节线粒体外膜的凋亡事件中的作用。
Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic Bcl-2 proteins have been shown to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of antiapoptotic Bcl-x(L) may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking, we obtained direct evidence for binding of Bcl-x(L) to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-x(L) was characterized by NMR with chemical shift perturbation and transferred cross-saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-x(L) that was found to insert into detergent micelles. Our results suggest that Bcl-x(L) can bind to one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-x(L) complex offers initial structural insight into the role of antiapoptotic Bcl-x(L) in regulating apoptotic events in the mitochondrial outer membrane.