Structural and mutational analysis of the ribosome-arresting human XBP1u
- Ludwig Maximilian University of Munich, Germany
- Stockholm University, Sweden
- Nara Institute of Science and Technology, Japan
Abstract
XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
Data availability
Generated models have been deposited in Protein Data Bank (PDB), and accessible via the following accession codes: 6R5Q, 6R6P, 6R6G and 6R7Q. Corresponding maps have been deposited in Electron Microscopy Data Bank (EMDB) and accessible with the following accession codes: EMDB-4729, EMDB-4737, EMDB-4735 and EMDB-4745.
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Structure of XBP1u-paused ribosome nascent chain complex with Sec61Protein Data Bank, 6R7Q.
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Structure of XBP1u-paused ribosome nascent chain complex with SRPProtein Data Bank, 6R6G.
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Structure of XBP1u-paused ribosome nascent chain complex (post-state)Protein Data Bank, 6R5Q.
Article and author information
Author details
Katharina Braunger
Funding
Deutsche Forschungsgemeinschaft (QBM (Quantitative Biosciences Munich) Graduate School Fellowship)
- Vivekanandan Shanmuganathan
Knut och Alice Wallenbergs Stiftelse (2012.0282)
- Gunnar von Heijne
Vetenskapsrådet (621-2014-3713)
- Gunnar von Heijne
Cancerfonden (15 0888)
- Gunnar von Heijne
Japan Society for the Promotion of Science (JP26116006)
- Kenji Kohno
Deutsche Forschungsgemeinschaft (GRK1721)
- Roland Beckmann
Boehringer Ingelheim Fonds (Graduate Fellowship)
- Katharina Braunger
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Shanmuganathan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structural and mutational analysis of the ribosome-arresting human XBP1u
eLife 8:e46267.
https://doi.org/10.7554/eLife.46267
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