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InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1–SMG8–SMG9 complex

Nature Structural & Molecular Biology volume 26, pages 1089–1093 (2019)Cite this article

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Abstract

We report the 3.45-Å resolution cryo-EM structure of human SMG1–SMG8–SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.

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Fig. 1: Cryo-EM structure of human SMG1–SMG8–SMG9.
Fig. 2: Interactions between SMG1 and SMG8–SMG9.
Fig. 3: InsP6-binding site of SMG1 and mTOR.

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Data availability

Cryo-EM density maps are deposited in the Electron Microscopy Data Bank, under accession numbers EMD-10347 and EMD-10348. The atomic model is deposited in the PDB, under accession number 6SYT. Source data for Fig. 3 and Extended Data Fig. 7 are provided with the paper.

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Acknowledgements

We thank S. Suppmann and N. Nagaraj from the Max Planck Institute of Biochemistry Core Facilities for advice on protein expression and for data in Extended Data Fig. 1, D. Wartini for excellent support with mammalian cell culture and the Max Planck Institute of Biochemistry cryo-EM facility for microscope access. We also thank members of our group for comments and discussions, in particular L. Langer. This study was supported by grants from the Max Planck Gesellschaft and the German Research Foundation (grant nos. DFG SFB1035, GRK1721, SFB/TRR 237) to E.C. and a Boehringer Ingelheim Fonds fellowship to M.L.

Author information

Author notes

  1. Mike Strauss

    Present address: Department of Anatomy and Cell Biology, McGill University, Montreal, Québec, Canada

  2. These authors contributed equally: Yair Gat, Jan Michael Schuller.

Authors and Affiliations

  1. Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Munich, Germany

    Yair Gat, Jan Michael Schuller, Mahesh Lingaraju, Fabien Bonneau & Elena Conti

  2. Biochemistry Core Facility, Max Planck Institute of Biochemistry, Munich, Germany

    Elisabeth Weyher

  3. Cryo-EM Facility, Max Planck Institute of Biochemistry, Munich, Germany

    Mike Strauss

  4. Immunoregulation Group, Max Planck Institute of Biochemistry, Munich, Germany

    Peter J. Murray

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Contributions

J.M.S. and Y.G. expressed, purified, and prepared protein complex from mammalian cell lines established by M.L. and F.B. Cryo-EM data were collected by M.S. and processed by J.M.S. Y.G. built the atomic model and performed biochemical assays. E.W. conducted the MS experiments. P.J.M. contributed to planning mTOR experiments. Y.G., J.M.S. and E.C. analyzed the structure and wrote the manuscript.

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Correspondence to Elena Conti.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical characterization of recombinant SMG1-SMG8-SMG9.

Biochemical characterization of recombinant SMG1-SMG8-SMG9 complex (SMG1c). (a) Size-exclusion chromatography assay showing the formation of homogeneous SMG1-SMG8-SMG9 ternary complex. After expression in mammalian cells, the complex was purified by size-exclusion column (Superose 6 Increase 10/300 GL, void volume of 8.0 ml). Top panel: chromatography profile, with absorbance at 280 nm and 260 nm shown as blue and purple traces, respectively. Bottom panel: Coomassie-stained SDS-PAGE gel, with samples from each fraction that eluted between 8.5–16 ml; the SMG1c eluted between 13–15 ml. (b) Mass spectrometry (MS) analysis of purified SMG1. Graphical representation shows the result of an in-gel peptide mass fingerprinting experiment. Identified peptides are indicated as red dashes at the corresponding position in the SMG1 sequence (horizontal axis) and are plotted against their intensity, as detected by MS (vertical axis). The uniform distribution of detected peptides over the entire amino acid sequence indicates that the entire SMG1 polypeptide is present in the purified sample.

Extended Data Fig. 2 Cryogenic electron microscopy (Cryo-EM) data collection and analysis.

Cryogenic electron microscopy (Cryo-EM) data collection and analysis. (a) A representative cryo-EM micrograph collected on an FEI Titan Krios microscope, operated at 300 kV and equipped with a K2 Summit camera. (b) Representative reference-free 2D class averages. (c) Angular distribution of the particles used for the final round of refinement. (d) Local-resolution analysis of the SMG1c. Map shows the variation in local resolution, as estimated by RELION. (e) Local-resolution analysis of the SMG1c (including the C-terminal region of SMG8). Map shows the variation in local resolution, as estimated by cryoSPARC. (f) 3D FSC and preferred orientation analysis of the dataset with the red line representing the estimated global FSC of 3.45 Å ± 1 SD (green dashed lines). A sphericity of 0.943 indicates a mostly isotropic map without preferred orientation bias. (g) Model vs. map FSC for the final PHENIX real-space refined model. (h) The model was probed for over-fitting by randomly perturbing the atoms by 0.5 Å and refining against the first of the two independent half-maps (work half-map, red). The resulting refined model was then used to calculate a model-map FSC against the second half-map (test half-map, green), which was not used for refinement. FSC-work and FSC-test curves show excellent agreement over the entire resolution range, validating the entire structure against over-fitting.

Extended Data Fig. 3 Cryo-EM data processing scheme.

Cryo-EM data processing scheme. The 849,831 particles from the 2D classification were initially 3D-classified into five classes. The two classes containing the best aligning particles were combined and auto-refined in RELION to a resolution of 3.45 Å. The dataset was further classified for the C-terminus of the SMG8 protein in cryoSPARC, using heterogeneous refinement. A class-showing density for the SMG8 C-terminal region was then selected and refined using homogenous refinement to a resolution of 3.57 Å.

Extended Data Fig. 4 Model fit and Ramachandran plot.

Quality of the structural model built de novo in the cryo-EM map. (a) Representative regions of the SMG1c and surrounding electron density are shown. (b) Ramachandran plot of the main-chain φ, and ψ conformational angles of the SMG1-SMG8-SMG9 atomic model. Areas of favored φ and ψ combinations are defined in dark blue (see also Supplementary Table 1).

Extended Data Fig. 5 G-fold protein regulators of SMG1 and mTOR.

G-fold protein regulators of the cytoplasmic PIKK proteins, SMG1 and mTOR. Structures of SMG1 bound to the G-domain proteins, SMG8 and SMG9 (a), and of mTOR bound to RHEB (PDB, code 6BCU) (b) are shown in similar orientations after superimposition of the kinase domains of SMG1 and mTOR. Although the overall binding site is similar, there are major differences. First, RHEB is a bona fide GTPase, whereas the G-domain regulators of SMG1 binds ATP rather than GTP, and may well lack catalytic activity. Second, RHEB is positioned with its GTP domain roughly facing the catalytic cleft of mTOR, in an opposite orientation to that of SMG9. Unlike SMG9, RHEB binds to HEAT repeats proximal to the catalytic head (‘bridge’), as well as the very N-terminal HEAT repeats of mTOR (‘horn’). Significantly, the ‘bridge’ region of the mTOR HEAT repeats forms an extensive interface with the globular RHEB and largely replaces the equivalent position of SMG8 in the SMG1 structure.

Extended Data Fig. 6 SMG9 purifies bound to ATP.

SMG9 purifies bound to ATP. (a) Dimerization interface between the G-fold-like domains of SMG8 (cyan) and SMG9 (green), showing the electron density around the nucleotide and Mg ion bound in the SMG9 G-fold. (b) Coomassie-stained SDS gel of the human SMG8-SMG9 complex purified from HEK 293T cells. (c) Analysis of the purified SMG8-SMG9 dimer by ion-pair HPLC-mass spectrometry. Top: HPLC extracted-ion chromatogram (EIC) of SMG8-SMG9 dimer. Bottom: MS analysis of the peak fraction indicated above, with the mass corresponding to ATP labeled. Full-length, unprocessed gel for b is shown in Source Data.

Extended Data Fig. 7 Validation of IP6 identification and interactions.

Validation of IP6 identification and interactions. (a) The SMG1 complex was analyzed by ion pair HPLC-MS (top and middle panels). To validate the identity of the 658.85 Da mass, this ligand was fragmented and the daughter ions analyzed by a second MS (bottom panel). As expected, the masses of the fragmented ions reflect a loss of individual groups of phosphate and water. (b) The chemical formula, composition and the monoisotopic mass (observed and expected) of each daughter ion. (c) Diagram of the chemical environment of IP6 bound to SMG1, showing the distances between the IP6 phosphate oxygens and the amino groups of SMG1 lysine and arginine side chains measured from the atomic model. We note, however, that the precision of these measurements is limited by the resolution of the electron density map.

Source data

Extended Data Fig. 8 Structural comparison of the PIKK FAT domains.

Structural comparison of the FAT domains of SMG1 with that of the other PIKK family members. In this figure, all structures are shown in the same orientation after optimal manual superposition of their FAT domains (FAT in violet, catalytic domain in slate, N-terminal HEAT in yellow). (a) mTOR (mammalian target of rapamycin) is from the PDB, code 4JSV, ATM (ataxia telangiectasia-mutated) is from the PDB, code 5MP0, ATR (ataxia- and Rad3-related) is from the PDB, code 5X60, DNA-PK (DNA protein kinase) is from the PDB, code 5LUQ, and the related protein TRRAP (transformation/transcription domain-associated protein) is from the PDB, code 5OJS. (b) Superposition of the structures shown in (a), despite poor structural conservation of the position and orientation of individual helices, the overall cleft-like feature that in SMG1 binds IP6 appears to be conserved within the PIKK family. Comparing the contact potential of each protein (not shown) we observe that the surface of this cleft is distinctly positive in SMG1, mTOR, TRRAP and ATR. The contact potential of DNA-PK is not as positive, and ATM was not included in this comparison due to the low resolution of this region.

Supplementary information

Supplementary Information

Supplementary Table 1 and Note 1.

Reporting Summary

Source data

Source Data Fig. 3

Unprocessed gels and autoradiography images

Source Data Extended Data Fig. 7

Unprocessed gel from Extended Data Fig. 7

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Gat, Y., Schuller, J.M., Lingaraju, M. et al. InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1–SMG8–SMG9 complex. Nat Struct Mol Biol 26, 1089–1093 (2019). https://doi.org/10.1038/s41594-019-0342-7

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