Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative

Pauwels, E.; Shewakramani, N. R.; De Wijngaert, B.; Camps, A.; Provinciael, B.; Stroobants, J.; Kalies, K.-U.; Hartmann, E.; Maes, P.; Vermeire, K.; Das, K.

doi:10.1101/2022.09.28.509949

bioRxiv · 10.1101/2022.09.28.509949

Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative

Pauwels, E.·Shewakramani, N. R.·De Wijngaert, B.·Camps, A.·Provinciael, B.·Stroobants, J.·Kalies, K.-U.·Hartmann, E.·Maes, P.·Vermeire, K.·Das, K.

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Abstract

During co-translational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the ER membrane. Our cryo-EM structure of ribosome-Sec61 shows binding of an ordered heterotetrameric TRranslocon-Associated Protein (TRAP) complex, in which TRAP-{gamma} is anchored at two adjacent positions of 28S rRNA and interacts with ribosomal protein L38 and Sec61/{gamma}. Four transmembrane helices (TMHs) of TRAP-{gamma} cluster with one C-terminal helix of each , {beta}, and {delta} subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-/{beta}/{delta} core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the CADA 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. Short SummaryCryo-EM structures reveal TRAP binding to ribosome-Sec61 complex, and CK147 inhibiting Sec61 by arresting the plug helix inside the channel.

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Pauwels, E., Shewakramani, N. R., De Wijngaert, B., Camps, A., Provinciael, B., Stroobants, J., Kalies, K.-U., Hartmann, E., Maes, P., Vermeire, K., Das, K.. 2022-09-28. Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative. https://doi.org/10.1101/2022.09.28.509949

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