Molecular basis for membrane pore formation by Bax protein carboxyl terminus.
Molecular basis for membrane pore formation by Bax protein carboxyl terminus.
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Bax 蛋白羧基末端形成膜孔的分子基础。
DOI：
10.1021/bi301195f
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Khaled,AnnetteR
中科院分区：
文献类型：
--
作者：
Tatulian,SurenA;Garg,Pranav;Nemec,KathleenN;Chen,Bo;Khaled,AnnetteR
Bax蛋白在细胞凋亡时线粒体膜透化和细胞色素c释放中起关键作用。有令人信服的证据表明，Bax的C-末端肽具有很强的在脂质膜中形成孔的能力。此外，我们最近的数据表明，细胞内表达的肽易位到线粒体，并对癌细胞产生致死作用，确定Bax的C-末端肽作为一个潜在的细胞毒性剂。我们已经表明，Bax的C-末端20个氨基酸的延伸（CRAC-KK; VTIFVAGVLTASLTIWKKMG），以及两个突变体，其中两个赖氨酸被谷氨酸（CRAC-EE）或亮氨酸（CRAC-LL）取代，在脂质膜中形成相对较大的孔。通过偏振傅里叶变换红外光谱、圆二色谱和荧光实验对磷脂膜中重构的肽的孔结构进行了分析。这些肽在膜内呈现α/β型二级结构。β-链和α-螺旋相对于膜法线显著倾斜（30-60度）。色氨酸残基嵌入两性离子膜中，距离膜中心8-9 μ m。结合基于钙黄绿素从脂质囊泡释放动力学确定的孔化学计量学，这些结构约束允许构建孔模型，其中8个肽分子形成“α/β-环”结构，孔内径为20-22 μ m。这些结果确定了一个强大的亲膜活性的Bax的C-末端，并提出了一个新的机制，肽可以有效地包裹细胞膜。关于肽的孔形成机制的知识可以促进基于肽的疗法的开发，以杀死癌症或其他有害细胞，如细菌或真菌。
Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. There is compelling evidence that the C-terminal peptide of Bax has strong capability of forming pores in lipid membranes. Moreover, our recent data have indicated that the intracellularly expressed peptide translocates to the mitochondria and exerts lethal effect on cancer cells, identifying the C-terminal peptide of Bax as a potential cytotoxic agent. We have shown that the C-terminal 20-amino acid stretch of Bax (BaxC-KK; VTIFVAGVLTASLTIWKKMG), as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), form relatively large pores in lipid membranes. The pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an α/β-type secondary structure within membranes. Both β-strands and α-helices are significantly (by 30-60 degrees) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8-9 Å from membrane center. Membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not BaxC-EE. Combined with pore stoichiometry determined based on the kinetics of calcein release from lipid vesicles, these structural constraints allow construction of a model of the pore where eight peptide molecules form an “α/β-ring” structure with pore inner diameter of 20-22 Å. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi.