Structural and mutational analysis of the ribosome-arresting human XBP1u

Abstract
Data availability
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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.

The following data sets were generated

(2019) Structure of XBP1u-paused ribosome nascent chain complex (rotated state)
Protein Data Bank, 6R6P.

http://www.rcsb.org/structure/6R6P
(2019) Structure of XBP1u-paused ribosome nascent chain complex with Sec61
Protein Data Bank, 6R7Q.

http://www.rcsb.org/structure/6R7Q
(2019) Structure of XBP1u-paused ribosome nascent chain complex with SRP
Protein Data Bank, 6R6G.

http://www.rcsb.org/structure/6R6G
(2019) Structure of XBP1u-paused ribosome nascent chain complex (post-state)
Protein Data Bank, 6R5Q.

http://www.rcsb.org/structure/6R5Q
(2019) Structure of XBP1u-paused ribosome nascent chain complex (post-state)
EMBL-EBI, EMD-4729.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4729
(2019) Structure of XBP1u-paused ribosome nascent chain complex with SRP
EMBL-EBI, EMD-4735.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4735
(2019) Structure of XBP1u-paused ribosome nascent chain complex (rotated state)
EMBL-EBI, EMD-4737.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4737
(2019) Structure of XBP1u-paused ribosome nascent chain complex with Sec61
EMBL-EBI, EMD-4745.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4745

Article and author information

Author details

Vivekanandan Shanmuganathan

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

Competing interests
The authors declare that no competing interests exist.
Nina Schiller

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Competing interests
The authors declare that no competing interests exist.
Anastasia Magoulopoulou

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Competing interests
The authors declare that no competing interests exist.
Jingdong Cheng

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

Competing interests
The authors declare that no competing interests exist.
Katharina Braunger

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9067-2155
Florian Cymer

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Competing interests
The authors declare that no competing interests exist.
Otto Berninghausen

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

Competing interests
The authors declare that no competing interests exist.
Birgitta Beatrix

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

Competing interests
The authors declare that no competing interests exist.
Kenji Kohno

Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Japan

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3503-6551
Gunnar von Heijne

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

For correspondence
gunnar.von.heijne@dbb.su.se

Competing interests
The authors declare that no competing interests exist.
Roland Beckmann

Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany

For correspondence
beckmann@genzentrum.lmu.de

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-4291-3898

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

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.