Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins
Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins
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DOI：
10.1042/bj20112079
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发表时间：
2012-06-15
影响因子：
4.1
通讯作者：
Shoshan-Barmatz, Varda
中科院分区：
文献类型：
--
作者：
Geula, Shay;Ben-Hail, Danya;Shoshan-Barmatz, Varda
结构研究将VDACI（电压依赖性阴离子通道1）N-末端区域置于通道孔内。然而，生化和功能研究表明，N-末端结构域是细胞质暴露。在本研究中，VDAC 1 N-末端结构域的位置和易位，其在电压门控中的作用，并作为抗凋亡蛋白的目标，得到解决。定点诱变和半胱氨酸残基取代，连同巯基特异性交联剂，用于显示VDAC 1 N-末端区域存在于动态平衡中，位于孔内或暴露在β-桶外。使用一个单一的半胱氨酸残基轴承VDAC 1，我们表明，N-末端区域位于孔内。然而，相同的区域可以暴露在孔外，在那里它与第二个VDAC 1分子的N-末端结构域二聚化。当N-末端区域α-螺旋结构被扰动时，分子内交联被废除，二聚化被增强。该突变体还显示降低的电压门控和降低的己糖激酶结合，但不显示抗凋亡蛋白Bcl-2和Bel-xL结合。取代甘氨酸残基的N-末端结构域GRS（富含甘氨酸的序列）产生较少的分子内交联的产品，但更多的二聚化，这表明GRS提供的N-末端易位所需的灵活性从内部孔的通道面。因此，N-末端迁移率可能有助于通道门控和与抗凋亡蛋白的相互作用。
Structural studies place the VDACI (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for antiapoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the beta-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region alpha-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bel-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.