How allosteric effectors influence protein translocation mediated by the Sec61 complex

变构效应子如何影响 Sec61 复合物介导的蛋白质易位

• 批准号: 434188682
• 负责人: Professor Dr. Volkhard Helms
• 金额: --
• 依托单位: Zentrum für Bioinformatik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/434188682?language=en
• 关键词: How; allosteric; effectors; influence; protein

In eukaryotes, the protein biosynthesis is carried out either by cytosolic ribosomes or by ribosomes that are attached to the membrane of the endoplasmatic reticulum (ER). In the latter case, the newly synthesized “nascent peptide chain” (NC) is typically translocated into the ER or laterally inserted into the ER membrane via an integral membrane protein complex. In eukaryotes, its central component is termed the Sec61 complex. Proteins that should be translocated across the ER membrane carry a "signal Peptide" (SP) at their N-terminus. In the ER, this sequence is then cleaved off by the enzyme signal peptidase. Upon exiting from the ribosome, the signal recognition particle (SRP) binds SP of the newly synthesized NC at the ribosome. Subsequently, upon interaction of SRP and SRP receptor at the ER membrane, SRP dissociates from SP which then is free to insert into the Sec61 channel. This pathway is termed the co-translational, SRP-dependent pathway, which is the focus of the proposed work. Not all NCs that should pass Sec61 may do this on their own. Certain NCs require the presence of additional accessory membrane proteins such as TRAP and Sec62-Sec63. Until recently, it was unclear which features of SPs guide them through different translocation pathways. In prior published work, we have contributed to unraveling physico chemical features of SPs that distinguish TRAP clients or Sec62-63 clients from those proteins that do not require aid by such accessory proteins. Still, the mechanistic role of these accessory proteins and how they mechanistically affect cotranslational translocation is unknown. Furthermore, several small molecule effectors have been described in the literature (e.g. eeyarestatins, mycolactone, and Ipomoeassin F) that modulate the protein translocation efficiency of the Sec61 complex. In prior published work, we combined ligand docking with data from electrophysiology to suggest how eeyarestatins derivatives bind differentially in the Sec61 pore. Based on our published findings and on recent advances in structure determination of the Sec61 complex, we now aim at unravelling the mechanistic details how allosteric effectors influence protein translocation mediated by the Sec61 complex. We will implement an integrated computational and experimental approach. Specific aims of the proposed project are: (i) we want to characterize how SPs bind in the Sec61 pore, (ii) we want to characterize how accessory proteins alter the conformational sampling of SP in the Sec61 pore, (iii) we want to characterize the molecular consequences upon binding of small-molecule effectors to Sec61.

在真核生物中，蛋白质的生物合成是通过细胞质核糖体或附着在内质网（ER）膜上的核糖体进行的。在后一种情况下，新合成的“新生肽链”（NC）通常易位到内质网或通过完整的膜蛋白复合物横向插入内质网膜。在真核生物中，其核心成分被称为Sec61复合物。应通过内质网膜转运的蛋白质在其n端携带一个“信号肽”（SP）。在内质网中，这个序列随后被酶信号肽酶切断。信号识别粒子（SRP）离开核糖体后，在核糖体上结合新合成的NC的SP。随后，在SRP与ER膜上的SRP受体相互作用后，SRP与SP分离，然后SP自由地插入Sec61通道。这种途径被称为共翻译，srp依赖的途径，这是提出的工作的重点。并不是所有应该通过Sec61的国家都可以自己这样做。某些NCs需要存在额外的辅助膜蛋白，如TRAP和Sec62-Sec63。直到最近，还不清楚SPs的哪些特征引导它们通过不同的易位途径。在之前发表的工作中，我们已经揭示了SPs的物理化学特征，这些特征区分了TRAP客户端或Sec62-63客户端与那些不需要这些辅助蛋白帮助的蛋白质。然而，这些辅助蛋白的机制作用以及它们如何影响共翻译易位的机制尚不清楚。此外，文献中已经描述了几种小分子效应物（例如，eyare他汀类药物、霉菌内酯和ipomoeasin F），它们调节Sec61复合物的蛋白质易位效率。在之前发表的工作中，我们将配体对接与电生理学数据相结合，以表明eeyare他汀类药物衍生物在Sec61孔中的结合差异。基于我们已发表的研究结果和最近在Sec61复合体结构确定方面的进展，我们现在的目标是揭示变构效应物如何影响由Sec61复合体介导的蛋白质易位的机制细节。我们将实施一个综合的计算和实验方法。拟议项目的具体目标是：(i)我们想要表征SPs如何在Sec61孔中结合，（ii）我们想要表征辅助蛋白如何改变Sec61孔中SP的构象采样，（iii）我们想要表征小分子效应物与Sec61结合后的分子后果。