Department of Microbiology, Immunology, and Transplantation, KU Leuven, Leuven 3000, Belgium.
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Leuven 3000, Belgium.
Sci Adv. 2023 Mar 3;9(9):eadf0797. doi: 10.1126/sciadv.adf0797.
During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28 ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the cyclotriazadisulfonamide derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147 resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.
在共翻译易位过程中，新生肽链的信号肽与 Sec61 易位体结合，启动蛋白质通过内质网膜的转运。我们的核糖体- Sec61 冷冻电镜结构显示，有序的异四聚体易位相关蛋白（TRAP）复合物结合，其中 TRAP-γ 锚定在核糖体 RNA 的两个相邻位置 28 并与核糖体蛋白 L38 和 Sec61α/γ 相互作用。TRAP-γ 的四个跨膜螺旋（TMH）与每个 α、β 和 δ 亚基的一个 C 端螺旋聚集在一起。该七 TMH 束有助于将新月形的三聚体 TRAP-α/β/δ 核心定位在内质网腔中，面向 Sec61 通道。此外，我们的体外测定建立了环三氮杂二硫代甲酰胺衍生物 CK147 作为易位体抑制剂。核糖体- Sec61-CK147 的结构揭示了 CK147 结合通道并从腔侧与塞子螺旋相互作用。CK147 的抗性突变围绕抑制剂。这些结构有助于理解 TRAP 功能，并为设计易位体抑制剂提供了一个新的 Sec61 位点。
