Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes.
Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes.
复制标题
代表志贺毒素 A1 结构域 C 末端的合成肽在磷脂膜中的插入和定向。
作者：
M. Saleh;J. Ferguson;J. Boggs;J. Gariépy
志贺毒素是一种由1个a亚基和5个B亚基组成的细菌蛋白。其A链具有一个蛋白酶敏感环（Cys-242-Cys-261），该环被裂解产生酶活性A1结构域和与B亚基五聚体相关的A2片段。所提出的毒素的作用方式与其逆行运输到内质网管，然后将其催化A1链转位到内质网膜的细胞质侧有关。构成A1链c端的信号序列样结构域（残基220-246）位于蛋白酶敏感环（残基247-258）内包含已知和假定的切割位点的区域之前。化学合成了与该c端对应的两个肽（残基220-246），以研究该信号序列样结构域是否可以与膜相互作用。这一特性可能为A1结构域跨ER膜的易位机制提供线索。第一肽代表天然序列，其中包括242位天然存在的半胱氨酸，并提供用于附着自旋标签的硫醇片段。第二个肽被设计为包含位于序列疏水核心的单个色氨酸残基（Ile232Trp），作为固有荧光探针。通过圆二色性、荧光和EPR光谱分析了两种肽与脂质囊泡的相互作用。肽在水缓冲液中缺乏结构，当与带负电荷的脂质囊泡结合时采用α -螺旋几何结构。在含色氨酸肽的溶液中加入脂质囊泡会导致其荧光最大值波长发生蓝移，并在335 nm处荧光强度增加，这表明该A1肽的疏水核心迁移到非极性环境中。在Cys-242上引入的一种羟基标记的肽类似物的EPR测量表明，在脂质囊泡存在的情况下，羟基探针的一部分迁移率降低。在pH为7时，膜结合探针被困在囊泡内的抗坏血酸完全还原，而被囊泡外添加的抗坏血酸仅部分还原，这表明肽的c端区域穿过膜双分子层或重新定位到靠近其内脂小叶表面的地方。最后，肽被显示插入脂质囊泡，导致钙黄蛋白以高肽脂比释放。这些结果表明A1链的c端尾部可能将该结构域锚定在内质网膜上。
Shiga toxin is a bacterial protein composed of one A and five B subunits. Its A chain possesses a protease sensitive loop (Cys-242-Cys-261) that is cleaved to produce an enzymatically active A1 domain and an A2 fragment associated with its B subunit pentamer. The proposed mode of action of the toxin is linked to its retrograde transport to the ER lumen followed by the translocation of its catalytic A1 chain to the cytoplasmic side of the ER membrane. A signal sequence-like domain (residues 220-246) which constitutes the C-terminus of the A1 chain precedes a region within the protease sensitive loop (residues 247-258) that contains known and putative cleavage sites. Two peptides corresponding to this C-terminus (residues 220-246) were chemically synthesized to investigate if this signal sequence-like domain can interact with membranes. Such a property may provide a clue to the mechanism of translocation of the A1 domain across the ER membrane. The first peptide represented the native sequence, which includes a naturally occurring cysteine at position 242 and provided a thiol moiety for the attachment of a spinlabel. A second peptide was designed to contain a single tryptophan residue (Ile232Trp) located within the hydrophobic core of the sequence which served as an intrinsic fluorescence probe. The interactions of both peptides with lipid vesicles were analyzed by circular dichroism, fluorescence, and EPR spectroscopy. The peptides lack structure in aqueous buffers and adopted an alpha-helical geometry when bound to negatively charged lipid vesicles. The addition of lipid vesicles to a solution of the tryptophan-containing peptide results in a blue shift in the wavelength of its fluorescence maxima as well as an increase in fluorescence intensity at 335 nm, suggesting that the hydrophobic core of this A1 peptide relocated to a nonpolar environment. EPR measurements of a proxyl-labeled analog of the peptide (introduced at Cys-242) indicated a decreased mobility of a fraction of the proxyl probe in the presence of lipid vesicles. At pH 7, the membrane-bound probe was completely reduced by ascorbate trapped inside vesicles but only partially reduced by ascorbate added outside the vesicles, suggesting that the C-terminal region of the peptide traversed the membrane bilayer or relocated close to the surface of its inner lipid leaflet. Finally, the peptide was shown to insert into lipid vesicles, causing the release of calcein at a high peptide:lipid ratio. These results suggest that the C-terminal tail of the A1 chain may anchor this domain into the ER membrane.