1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Structural and mutational analysis of the ribosome-arresting human XBP1u

  1. Vivekanandan Shanmuganathan
  2. Nina Schiller
  3. Anastasia Magoulopoulou
  4. Jingdong Cheng
  5. Katharina Braunger
  6. Florian Cymer
  7. Otto Berninghausen
  8. Birgitta Beatrix
  9. Kenji Kohno
  10. Gunnar von Heijne  Is a corresponding author
  11. Roland Beckmann  Is a corresponding author
  1. Ludwig-Maximilians-Universität München, Germany
  2. Stockholm University, Sweden
  3. Nara Institute of Science and Technology, Japan
  4. Science for Life Laboratory, Stockholm University, Sweden
Research Article
Cite this article as: eLife 2019;8:e46267 doi: 10.7554/eLife.46267
7 figures, 8 data sets and 3 additional files

Figures

Schematic representation of the IRE1α-XBP1u pathway mediating UPR.

Interaction of the XBP1u nascent chain with the ribosomal exit tunnel leads to translational pausing, resulting in SRP recruitment to the RNC, followed by targeting to Sec61 on the ER membrane. IRE1α localized near Sec61 during ER stress can splice XBP1u mRNA to XBP1s mRNA, which acts as transcription factor in alleviating ER stress.

https://doi.org/10.7554/eLife.46267.002
Figure 2 with 3 supplements
Structural analysis of XBP1u mediated ribosomal pausing.

(A) Schematic representation of the XBP1u-del-HR1-mu construct used for purification. The construct encodes N-ter (8X-His, 3X-Flag tag and 3C-protease site), hydrophobic region 2 (red), AP (green) and C-ter (HA-tag). Model for nascent chain in the tunnel, and P-site and A-site positions were denoted as well. (B) Transverse section of cryo-EM structure of the paused XBP1u-RNC showing the peptidyl-tRNA (green) with small and large subunits colored in yellow and gray, respectively. Densities for nascent chain, small and large subunit are displayed at contour levels of 1.5, 1.7 and 4.1 σ, respectively. (C) Close-up views showing the two tRNA states of the XBP1u-RNC, post (top panel) and rotated (bottom panel). For the post state (top panel), P- and E-site tRNA are displayed at 3.4 and 3 σ. While small and large subunit densities are shown at 2.7 and 2.8 σ, respectively. For the rotated state (bottom panel), A/P-, P/E-tRNA, large and small subunit densities are shown at 2.6, 3.1, 3.1 and 3.3 σ, respectively. (D) Overview of the XBP1u nascent chain in the ribosomal tunnel. Surface representation of the electron density: nascent chain (green), uL4 (orange), uL22 (blue) and ribosomal tunnel (gray). Densities for nascent chain, large subunit, uL4 and uL22 are displayed at the contour levels of 2.6, 3.9, 3.2 and 4.1 σ, respectively.

https://doi.org/10.7554/eLife.46267.003
Figure 2—figure supplement 1
Cryo-EM data processing of the XBP1u nascent chain stalled ribosomes.

In silico sorting procedure of the data is shown in the schema. Intermediate densities are shown with 60S in blue, 40S in yellow and tRNAs in green. For details check experimental methods section for data processing.

https://doi.org/10.7554/eLife.46267.004
Figure 2—figure supplement 2
Resolution of XBP1u-RNCs.

(Left panel) (A–B) Traverse section of post and rotated state XBP1u-RNC final map colored according to local resolution are shown here. Relion generated local resolution filtered maps are used. (C–D) Electron density maps of XBP1u-RNC with SRP and Sec61 colored according to local resolution. Lowpass filtered maps at 6 Å are used in this figure. Right panel (A–D) Fourier shell correlation (black) curve of the final map, indicating average resolutions (FSC = 0.143, dashed black line). FSC curves calculated between final map and model (orange), as well as the self (green) and cross validated (brown) correlation curves for respective XBP1u-RNC states are plotted, indicating resolutions (FSC = 0.5 Cref, dashed blue line).

https://doi.org/10.7554/eLife.46267.005
Figure 2—figure supplement 3
XBP1u nascent chain resolution in the ribosomal tunnel and comparison to other known stalling peptides.

(A–B) Isolated XBP1u nascent chain density of the post and rotated state XBP1u-RNC colored by local resolution. Post and rotated state nascent chain densities are displayed at the contour level of 3 σ. (C–G) Superposition of XBP1u nascent chain (model in forest green, surface in light green) with Sec61b (orange, PDB ID 3JAG) (Voorhees and Hegde, 2015), hCMV (pink, PDB ID 5A8L) (Matheisl et al., 2015), MifM (purple, PDB ID 3J9W) (Sohmen et al., 2015) and VemP (blue, PDB ID 5NWY) (Su et al., 2017) respectively.

https://doi.org/10.7554/eLife.46267.006
Stabilizing interactions of XBP1u nascent chain with the ribosomal exit tunnel.

On the left shown nascent chain model (green) with density (gray mesh), and some interacting 28S rRNA bases and ribosomal protein residues are shown. Isolated nascent chain density is displayed at contour level of 1.28 σ. (A) Lys257 of XBP1u (green) is at the hydrogen bond making distance internally within XBP1u residue Arg251. (B) Trp249 of the XBP1u stacks internally onto Gln248. (C) Met260 of XBP1u makes a hydrophobic interaction with U4531 of 28S rRNA (blue). (D) Lys257 stacking with the base U4532 (E) Trp256 and Trp249 of XBP1u displace a ribosomal tunnel base G3904 (blue). G3904 conformation with XBP1u is compared with didemnin B treated ribosome (cyan, PDB ID 5LZS). (F) Arg251 of XBP1u makes a salt-bridge interaction with the exit tunnel base A4388. (I, G) Gly250 and Gln253 of XBP1u are in the distance for making hydrogen bond interaction with 28S rRNA bases A3908 and U4555. (H) Tyr241 of XBP1u stacks onto C2794. (J–L) Constriction site protein residues making interaction with XBP1u are shown: uL4 (orange) and uL22 (pink).

https://doi.org/10.7554/eLife.46267.007
Figure 4 with 1 supplement
Silencing of peptidyl transferase activity by XBP1u nascent chain.

(A) Conformation of C4398 in XBP1u-RNC (blue). (B) C4398 conformation in the paused complex in comparison with A-site accommodated 80S (PDB ID 5GAK, softblue) and with a post state 80S without an A-site tRNA (PDB ID 5AJ0, softpink) (C) Model of an incoming A-site tRNA (yellow, PDB ID 5GAK) clashes with Leu259 of XBP1u. Accommodation of A-site tRNA is prevented by XBP1u.

https://doi.org/10.7554/eLife.46267.008
Figure 4—figure supplement 1
Comparison of C4398 (C2452) and U4531 (U2585) conformation in XBP1u-RNC with other post-state ribosome 80S models.

(A) State of the base C4398 and U4531 in XBP1u-RNC (blue). (B) State of C4398 and U4531 compared with post state 80S (PDB ID 5AJ0, softpink and 5LZS, softblue) without an accommodated A-site tRNA. (C) - (D) (A) and (B) displayed with isolated density for the base C4398 (XBP1u-RNC in blue, EMD ID 2875, softpink and 4130, softblue).

https://doi.org/10.7554/eLife.46267.009
Figure 5 with 1 supplement
Cryo-EM structures of XBP1u-RNC with SRP and Sec61.

(A) Cryo-EM reconstruction of XBP1u-RNC with SRP: small (yellow), large (gray), SRP (orange) and hydrophobic region 2 of XBP1u (purple). Same view, a transverse section is shown with XBP1u nascent chain and P-site tRNA (green). Densities of SRP, small and large subunit, and the nascent chain are shown at the contour levels of 2.2, 4, 3.1 and 3.1 σ, respectively. (B) Close-up view of SRP54 M-domain with a top and cross sectional view showing HR2 of XBP1u. (C) Sec61 bound to paused XBP1u-RNC. Cross sectional view: Sec61 (blue), small and large ribosomal subunits, and nascent chain density shown. Idle Sec61 model (cyan, PDB ID 3J7Q) and Sec61 model bound to XBP1u-RNC (blue). Lateral gate is highlighted in both models (purple). (D) Unaltered nascent chain in three different states: RNC alone (green), RNC with SRP (orange) and RNC with Sec61 (blue). Density of the nascent chain also colored respectively. From left to right: nascent chain densities are displayed at following contour levels: 1.6, 1.2, 2.7 and 1 σ, respectively.

https://doi.org/10.7554/eLife.46267.010
Figure 5—figure supplement 1
In silico sorting and local resolution.

(A–B) Cryo-EM data processing of XBP1u-RNC-SRP and XBP1u-RNC-Sec61. In silico sorting of both the datasets is schematically shown. (C–D) Isolated densities of Sec61 and SRP are colored according to their local resolution.

https://doi.org/10.7554/eLife.46267.011
Figure 6 with 2 supplements
Force profile measurement and saturation mutagenesis of the XBP1u AP.

(A) Construct used for mutagenesis screens. Y indicates the acceptor site for N-linked glycosylation. The amino acid sequence of the H segment and its flanking GGPG….GPGG residues is shown below. SDS-PAGE gel analysis of a full-length control (FLc, arrest-inactivating mutant), a construct with a Q242W mutation, and an arrest control (Ac) with stop codon immediately downstream of the AP. Full-length species are indicated by circles and arrested species by squares. Black and white colors indicate glycosylated and non-glycosylated species, as shown by Endo H digestion. (B) Force profiles measured for LepB-XBP1u (S255A) (red curve) and LepB-XBP1u (S255A, P254C) (blue curve) by in vitro translation in RRL supplemented with dog pancreas rough microsomes. A force profile measured in the E. coli-derived PURE in vitro translation system for the same construct but with the SecM(Ms) AP (Ismail et al., 2012) is included for comparison (gray curve). (C) Saturation mutagenesis of LepB-XBP1u (S255A, L = 43). Residues 241–262 were mutated to all 19 other natural amino acids and fFL values were determined. Residues are color-coded as follows: hydrophobic (orange), polar (blue), basic (green), acidic (brown), and G, P and C (purple). (D) Same as in C, but for LepB-XBP1u (P254V, S255A, L = 43). (E) Logo plot of the XBP1u AP, based on 90 homologous BLAST hits. The total height of each column reflects the sequence conservation in that position, and residue frequencies at a given position are indicated by the relative height of each residue in the column. The sequence variants found in the natural sequences are shown on the right.

https://doi.org/10.7554/eLife.46267.012
Figure 6—source data 1

Source for Figure 6 force profile experiments.

https://doi.org/10.7554/eLife.46267.015
Figure 6—figure supplement 1
LepB-XBP1u constructs.

Amino acid sequences of the LepB-XBP1u[L = 43] constructs used in the mutagenesis scans.

https://doi.org/10.7554/eLife.46267.013
Figure 6—figure supplement 2
Analysis of Cys positioning by cross-linking.

(A) No high-Mw disulfide-bonded crosslinked product is seen when in-vitro translated LepB-XBP1u[P254C, S255A; L = 43] construct is analyzed by non-reducing SDS-PAGE in the absence or presence of DTT. (B) fFL is reduced slightly when C247 is mutated to S, from 0.27 for LepB-XBP1u[P254C, S255A; L = 43] to 0.14 for LepB-XBP1u[C247S, P254C, S255A; L = 43] (n = 3). FL: full length form; A: arrested form.

https://doi.org/10.7554/eLife.46267.014
Schematic representation of the XBP1u pausing motif in the exit tunnel.

XBP1u residues color coded for number residues potency based on mutagenesis data. Turn formed by XBP1u is highlighted by a light blue box. Inset shows a schematic model of the PTC summarizing the pausing mechanism.

https://doi.org/10.7554/eLife.46267.016

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: 4729, 4737, 4735 and 4745.

The following data sets were generated
  1. 1
    Electron Microscopy Data Bank
    1. Shanmuganathan Vivekanandan
    2. Cheng Jingdong
    3. Braunger Katharina
    4. Berninghausen Otto
    5. Beatrix Birgitta
    6. Beckmann Roland
    (2019)
    ID EMD-4745. Structure of XBP1u-paused ribosome nascent chain complex with Sec61.
  2. 2
    Protein Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. Braunger Katharina
    4. Berninghausen Otto
    5. Beatrix Birgitta
    6. Beckmann Roland
    (2019)
    ID 6R7Q. Structure of XBP1u-paused ribosome nascent chain complex with Sec61.
  3. 3
    Protein Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. Braunger Katharina
    4. Berninghausen Otto
    5. Beatrix Birgitta
    6. Beckmann Roland
    (2019)
    ID 6R6G. Structure of XBP1u-paused ribosome nascent chain complex with SRP.
  4. 4
    Electron Microscopy Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. Braunger Katharina
    4. Berninghausen Otto
    5. Beckmann Roland
    (2019)
    ID EMD-4735. Structure of XBP1u-paused ribosome nascent chain complex with SRP.
  5. 5
    Protein Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. O Berninghausen
    4. R Beckmann
    (2019)
    ID 6R6P. Structure of XBP1u-paused ribosome nascent chain complex (rotated state).
  6. 6
    Protein Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. O Berninghausen
    4. R Beckmann
    (2019)
    ID 6R5Q. Structure of XBP1u-paused ribosome nascent chain complex (post-state).
  7. 7
    Electron Microscopy Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. O Berninghausen
    4. R Beckmann
    (2019)
    ID EMD-4729. Structure of XBP1u-paused ribosome nascent chain complex (post-state).
  8. 8
    Electron Microscopy Data Bank
    1. V Shanmuganathan
    2. J Cheng
    3. O Berninghausen
    4. R Beckmann
    (2019)
    ID EMD-4737. Structure of XBP1u-paused ribosome nascent chain complex (rotated state).

Additional files

Supplementary file 1

Cryo-EM data collection, refinement and validation statistics.

Summary of parameters related cryo-EM data collection and processing.

https://doi.org/10.7554/eLife.46267.017
Supplementary file 2

List of aminoacid sequences representing the constructs used in Figure 6B.

Representing red curve (mutation S255A in the XBP1u AP) and blue curve (mutations S255A and P254C in the XBP1u AP) in Figure 6B.

https://doi.org/10.7554/eLife.46267.018
Transparent reporting form
https://doi.org/10.7554/eLife.46267.019

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