Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: Forces involved and implication for inactivation of membrane proteins
Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: Forces involved and implication for inactivation of membrane proteins
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DOI：
10.1016/j.jmb.2004.09.066
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发表时间：
2004-11-26
影响因子：
5.6
通讯作者：
Shai, Y
中科院分区：
文献类型：
--
作者：
Sal-Man, N;Gerber, D;Shai, Y
膜内的蛋白质-蛋白质相互作用，部分或全部由跨膜（TM）结构域介导，参与许多重要的细胞过程。由于膜环境的独特特征使得蛋白质-蛋白质组装成为可能，而蛋白质-蛋白质组装在溶液中不具有能量优势，因此可溶性蛋白质组装中涉及的结构限制对于TM结构域的组装不一定有效。在这里，我们使用的N-末端TM结构域（焦油-1）的大肠杆菌天冬氨酸受体作为模型系统，用于检查立体特异性的TM-TM相互作用在体外和体内的隔离系统，并在上下文中的完整的受体。为此，我们合成了Tar-1全L和全D氨基酸TM肽、突变TM肽和无关TM肽。数据显示：（ii）Tar-1all-L和all-D，但不是对照肽，在细菌ToxR组装系统中对Tar-1 TM同源二聚化表现出剂量依赖性显性负效应，表明野生型样相互作用;最有趣的是，（iii）Tar-1all-L和all-D都显示出在体内抑制全长受体的趋化反应的显著能力。通过共聚焦成像证实了肽与细菌的结合，并且Western印迹证实了ToxR Tar-1嵌合体蛋白水平不受外源肽的存在的影响。这些研究结果提出了第一个证据表明，一个全D TM域肽在体内的行为类似于其父母的所有-L肽，并表明TM域的二聚化主要是由侧链相互作用介导的，而不是几何拟合构象。此外，该研究为利用无蛋白水解肽修饰膜蛋白的功能提供了新的途径。(C)2004爱思唯尔有限公司保留所有权利。
Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-L and all-D amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-D specifically associated with Tar-1 all-L within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-L and all-D, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-L and all-D showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-D TM domain peptide acts in vivo similarly to its parental all-L peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides. (C) 2004 Elsevier Ltd. All rights reserved.