Sal-Man Neta, Gerber Doron, Shai Yechiel
Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
J Mol Biol. 2004 Nov 26;344(3):855-64. doi: 10.1016/j.jmb.2004.09.066.
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.
膜内的蛋白质-蛋白质相互作用部分或完全由跨膜（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性能够实现蛋白质-蛋白质组装，而这种组装在溶液中在能量上并不占优势，所以可溶性蛋白质组装中涉及的结构限制不一定适用于TM结构域的组装。在这里，我们使用大肠杆菌天冬氨酸受体的N端TM结构域（Tar-1）作为模型系统，在体外和体内的分离系统以及完整受体的背景下研究TM-TM相互作用的立体特异性。为此，我们合成了Tar-1全L型和全D型氨基酸TM肽、一种突变TM肽和一种无关的TM肽。数据显示：（i）通过荧光能量转移实验确定，Tar-1全D型在模型脂质膜内与Tar-1全L型特异性结合；（ii）Tar-1全L型和全D型，而非对照肽，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的显性负效应，表明存在类似野生型的相互作用；最有趣的是，（iii）Tar-1全L型和全D型在体内均表现出显著抑制全长受体趋化反应的能力。通过共聚焦成像证实了肽与细菌的结合，蛋白质印迹证实外源性肽的存在不影响ToxR Tar-1嵌合蛋白水平。这些发现首次证明全D型TM结构域肽在体内的作用与其亲本全L型肽相似，并表明TM结构域的二聚化主要由侧链相互作用介导，而非几何拟合构象。此外，该研究为通过无蛋白水解的肽修饰膜蛋白功能提供了一种新方法。
