Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8
- Max Planck Institute of Biochemistry, Germany
Figures
Figure 1 with 2 supplements
SMG1i specifically inhibits SMG1 kinase activity in vitro.
(A) Structure of the SMG1 inhibitor (SMG1i). (B) Titration of SMG1i using a mass spectrometry-based phosphorylation assay with 500 nM SMG1-8-9 and the indicated UPF1-derived peptides as substrates. …
-
Figure 1—source data 1
Unedited images for gels shown in Figure 1.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig1-data1-v2.zip
Figure 1—figure supplement 1
Characterization of SMG1 inhibitor.
(A) Liquid chromatography-mass spectrometry (LC-MS) experiment with the SMG1 inhibitor sample used throughout this study. The expected mass for SMG1i is 566.13 Da. Differences of +1 are caused by …
Figure 1—figure supplement 2
Radioactivity-based phosphorylation assays using SMG1 inhibitor with SMG1 and mTOR.
(A) Titration of SMG1i using 100 nM mTOR-LST8 and GST-AKT1 as a substrate. The Coomassie-stained gel is shown on top and the radioactive signal on the bottom. (B, C) SMG1-8-9 or mTOR-LST8 kinase …
-
Figure 1—figure supplement 2—source data 1
Unedited images for gels shown in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig1-figsupp2-data1-v2.zip
Figure 2 with 3 supplements
Structural basis for selective targeting of SMG1 by the SMG1 inhibitor.
(A) Model of the SMG1-8-9 kinase complex bound to SMG1i. SMG1 is in gray, SMG8 is in blue, and SMG9 is shown in green. SMG1i is shown as a magenta model overlaid with the isolated transparent …
Figure 2—figure supplement 1
Resolution distribution and isotropy of SMG1-centered cryo-EM maps bound to SMG1i.
SMG1i-bound reconstructions used in this study for model building are colored according to estimated local resolution shown in two different orientations. A three-dimensional Fourier shell …
Figure 2—figure supplement 2
Cryo-EM data processing of SMG1i data set.
(A) 2D class averages of SMG1-8-9 and SMG1-9 are calculated from the final particle stacks. Scale bars≈100 Å. (B) Processing scheme. Processing steps are indicated in blue; particle numbers and …
Figure 2—figure supplement 3
Further details of SMG1i binding and specificity.
(A) Multiple sequence alignment of parts of the kinase domains (N- and C-lobe indicated) belonging to the catalytically active members of the PIKK family with residues colored by identity. Residues …
Figure 3 with 4 supplements
Structures of SMG1-8-9 and SMG1-9 complexes reveal that the SMG1 insertion domain can block the substrate-binding path in the presence of SMG8.
(A) Cryo-EM density of SMG1-8-9 bound to SMG1i. Density for the inhibitor is in magenta, the N-terminus of the SMG1 insertion is in red, and all other parts as indicated. (B) Cryo-EM density of …
Figure 3—figure supplement 1
Details of the SMG1 insertion N-terminus.
(A) Model of SMG1 active site bound to UPF1-LSQ substrate and AMPPNP (PDB identifier: 6Z3R) shown superimposed with the corresponding EM density (EMD-11063) and the densities for apo SMG1-8-9 …
Figure 3—figure supplement 2
Resolution distribution and isotropy of SMG1-centered cryo-EM maps bound to AMPPNP.
AMPPNP-bound reconstructions used in this study for model building are colored according to estimated local resolution shown in two different orientations. A three-dimensional FSC plot is included …
Figure 3—figure supplement 3
Cryo-EM data processing of AMPPNP data set.
Processing steps are indicated in blue; particle numbers and percentages with respect to initial candidate particles are shown for relevant classes. Colored, dashed rectangles indicate the different …
Figure 3—figure supplement 4
Details of the SMG1-9 complex.
(A) Overlay of SMG1 (PDB identifier: 6L53) and SMG1-9 detailing movements of the N-terminal HEAT repeats. A front and a side view are shown and binding sites for SMG8 and SMG9 are indicated by gray …
Figure 4 with 4 supplements
The SMG1 insertion domain can block overall access to the kinase active site.
(A) Cryo-EM map after 3D variability analysis filtered by resolution and segmented. Two different views displaying extra density for SMG8 C-terminus (dark blue) and SMG1 insertion domain. The …
-
Figure 4—source data 1
Table with cross-linking data used in Figure 4.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig4-data1-v2.xls
-
Figure 4—source data 2
Unedited images for gels shown in Figure 4.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig4-data2-v2.zip
Figure 4—figure supplement 1
Cross-linking mass spectrometry of SMG1-8-9.
(A) Two samples of SMG1-8-9 (lanes 1 and 2) were incubated with BS3 (lanes 3 and 4) and analyzed using SDS-PAGE and Coomassie staining. (B) Exemplary intra cross-links detected for SMG1 mapped on …
-
Figure 4—figure supplement 1—source data 1
Unedited images for gels shown in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Selected spectra of detected SMG1-8-9 intra-links.
(A–D) Cross-links are shown above each panel. All spectra showed good sequence coverage with full y-ion series, many b-ions, and highly specific fragments.
Figure 4—figure supplement 3
Integration of cryo-EM, cross-linking MS, and AlphaFold data reveals a model for the SMG8 C-terminus.
(A) AlphaFold model of full-length SMG8 (UniProt: Q8ND04) colored by per-residue confidence score (pLDDT) (Jumper et al., 2021). The previously unmodeled SMG8 C-terminus is indicated. (B) AlphaFold …
Figure 4—figure supplement 4
Further characterization of SMG1-8-9—centered interactions.
(A) Coomassie-stained SDS-PAGE analysis of pull-down experiment showing that the interaction between SMG1 insertion domain (SMG12427–3606) and SMG8 C-terminus (SMG8728–991) is dependent on low-salt …
-
Figure 4—figure supplement 4—source data 1
Unedited images for gels shown in Figure 4—figure supplement 4.
- https://cdn.elifesciences.org/articles/72353/elife-72353-fig4-figsupp4-data1-v2.zip
Figure 5
Hypothetical model of SMG1 kinase regulation.
Structural and biochemical data suggest different layers of regulation on SMG1 kinase activity. Upon ATP binding (orange), the SMG1 kinase adopts an autoinhibited conformation (step 1)—mediated by …
Author response image 1
SDS-PAGE analysis of complexes purified from cells transfected to stably express either TwinStrep-SMG1-8-9 or TwinStrep-SMG1-9, as indicated.
Note the co-purification of endogenous SMG8 from the cell line not transfected with SMG8.
Author response image 2
Residues of the SMG1 kinase domain (K2363, K2370) that cross-link to the SMG1 insertion C-terminus are observed in close proximity to the additional density (red).
Related to Figure 4 C.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | SMG1 | Shigeo Ohno lab | Uniprot Q96Q15 | |
| Gene (H. sapiens) | SMG8 | Shigeo Ohno lab | Uniprot Q8ND04 | |
| Gene (H. sapiens) | SMG9 | Shigeo Ohno lab | Uniprot Q9H0W8 | |
| Cell line (H. sapiens) | HEK293T | ATCC | ||
| Strain, strain background (Escherichia coli) | BL21 Star (DE3) pRARE | EMBL Heidelberg Core Facility | Electrocompetent cells | |
| Peptide, recombinant protein | UPF1-LSQ (peptide 1078) and derivative | In-house as described in doi: https://elifesciences.org/articles/57127 | ||
| Chemical compound, drug | SMG1 inhibitor | Robert Bridges, Rosalind Franklin University of Medicine and Science, and the Cystic Fibrosis Foundation | ||
| Chemical compound, drug | AMPPNP | Sigma-Aldrich | ||
| Chemical compound, drug | ATP | Sigma-Aldrich | ||
| Software, algorithm | SerialEM | https://bio3d.colorado.edu/SerialEM/ | ||
| Software, algorithm | Focus | https://focus.c-cina.unibas.ch/ wiki/doku.php | v1.1.0 | |
| Software, algorithm | RELION | doi: 10.7554/eLife.42166 | RELION 3.0 | |
| Software, algorithm | Cryosparc | doi: 10.1038/nmeth.4169 | Cryosparc2 | |
| Software, algorithm | UCSF Chimera | UCSF, https://www.cgl.ucsf.edu/chimera/ | ||
| Software, algorithm | UCSF ChimeraX | UCSF, https://www.rbvi.ucsf.edu/chimerax/ | ||
| Software, algorithm | COOT | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | ||
| Software, algorithm | PHENIX | https://www.phenix-online.org/ | PHENIX 1.17 | |
| Software, algorithm | Molprobity | Duke Biochemistry, http://molprobity.biochem.duke.edu/ | ||
| Software, algorithm | PyMOL | PyMOL Molecular Graphics System, Schrodinger LLC | PyMOL 2.3.2 |
Additional files
-
Supplementary file 1
Cryo-EM data collection, refinement and validation statistics.
- https://cdn.elifesciences.org/articles/72353/elife-72353-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/72353/elife-72353-transrepform1-v2.pdf
