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.
-
Structure of XBP1u-paused ribosome nascent chain complex with Sec61Protein Data Bank, 6R7Q.
-
Structure of XBP1u-paused ribosome nascent chain complex with SRPProtein Data Bank, 6R6G.
-
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.
Reviewing Editor
- Ramanujan S Hegde, MRC Laboratory of Molecular Biology, United Kingdom
Publication history
- Received: February 20, 2019
- Accepted: June 20, 2019
- Accepted Manuscript published: June 27, 2019 (version 1)
- Version of Record published: July 11, 2019 (version 2)
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.
Metrics
-
- 3,546
- Page views
-
- 512
- Downloads
-
- 26
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structural and mutational analysis of the ribosome-arresting human XBP1u
eLife 8:e46267.
https://doi.org/10.7554/eLife.46267
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.
