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Neddylation requires glycyl-tRNA synthetase to protect activated E2

Nature Structural & Molecular Biology volume 23, pages 730–737 (2016)Cite this article

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Abstract

Neddylation is a post-translational modification that controls the cell cycle and proliferation by conjugating the ubiquitin-like protein NEDD8 to specific targets. Here we report that glycyl-tRNA synthetase (GlyRS), an essential enzyme in protein synthesis, also plays a critical role in neddylation. In human cells, knockdown of GlyRS, but not knockdown of a different tRNA synthetase, decreased the global level of neddylation and caused cell-cycle abnormality. This function of GlyRS is achieved through direct interactions with multiple components of the neddylation pathway, including NEDD8, E1, and E2 (Ubc12). Using various structural and functional approaches, we show that GlyRS binds the APPBP1 subunit of E1 and captures and protects activated E2 (NEDD8-conjugated Ubc12) before the activated E2 reaches a downstream target. Therefore, GlyRS functions as a chaperone that critically supports neddylation. This function is probably conserved in all eukaryotic GlyRS enzymes and may contribute to the strong association of GlyRS with cancer progression.

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Figure 1: GlyRS binds NEDD8 and enhances neddylation.
Figure 2: GlyRS preferentially binds Ubc12N8.
Figure 3: GlyRS binds APPBP1 and captures newly released Ubc12N8.
Figure 4: GlyRS regulates the cell cycle by promoting cullin neddylation.
Figure 5: The active site is not essential to the role of GlyRS in neddylation.
Figure 6: Dual function of GlyRS in protein synthesis and neddylation.

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Acknowledgements

We thank B. Schulman (St. Jude Children's Research Hospital) and W. Harper (Harvard Medical School) for providing plasmids and advice on the project. We thank W. He and H. Zhou (The Scripps Laboratories for tRNA Synthetase Research) for providing plasmids of GlyRS. We thank P. Schimmel, M.H. Nawaz, and other members of The Scripps Laboratories for tRNA Synthetase Research for advice, discussion, and technical support. This research was supported by US National Institutes of Health grant R01GM088278 (X.-L.Y.).

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Authors and Affiliations

  1. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Zhongying Mo, Qian Zhang, Ze Liu, Yi Shi, Litao Sun & Xiang-Lei Yang

  2. Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

    Zhongying Mo, Qian Zhang, Ze Liu, Yi Shi, Litao Sun & Xiang-Lei Yang

  3. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida, USA

    Janelle Lauer & Patrick R Griffin

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Contributions

X.-L.Y. and Z.M. designed experiments, analyzed data, and wrote the manuscript. Z.M. performed the molecular cloning, binding analysis, structural docking, bioinformatic analysis, and additional biochemical analysis. Z.M. and Q.Z. carried out protein purification. Z.M. and Z.L. performed the cell-cycle analysis. J.L. and P.R.G. performed the HDX analysis. Y.S. and L.S. contributed to biochemical analysis.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the GlyRS-NEDD8 interaction.

(a) Hydrogen-deuterium exchange (HDX) analysis of GlyRS in the presence of NEDD8. Changes in deuterium incorporation resulting from the NEDD8 interaction are mapped to the sequence and the crystal structure of GlyRS (PDB 2PME). (b,c) Di-glycine motif of NEDD8 is not essential for GlyRS. Biolayer interferometry analysis comparing GlyRS binding to the full-length NEDD8 (b) and the truncated NEDD8 lacking the C-terminal di-glycine motif (NEDD8ΔGG) (c). His-tagged NEDD8 proteins were immobilized to the Ni-NTA sensor tips. Tag-free GlyRS (yellow green, 250 nM; orange,500 nM) were used to detect the interaction with NEDD8.

Supplementary Figure 2 GlyRS does not affect Ube2F conjugation.

(a) Western blot analysis to detect N8-conjugation of Ube2F in HeLa cells transfected with plasmids expressing shRNAs against GlyRS (shGlyRS) or SerRS (shSerRS) or a control vector. (b) Biolayer interferometry analysis to compare the binding of Ubc12 and Ube2F (both at 1.0 µM) to immobilized GST-GlyRS.

Supplementary Figure 3 The catalytic domain of GlyRS is the major binding site for Ubc12.

(a) In vitro neddylation assay with γ-32P-ATP labeled NEDD8. (b) In vitro neddylation assay using fluorescein modified NEDD8. (c) GST pull-down analysis to detect the interaction of GST-Ubc12 with GlyRS and its truncated fragments. (d) Hydrogen-deuterium exchange (HDX) analysis of GlyRS in the presence of Ubc12. Changes in deuterium incorporation resulting from the Ubc12 interaction are mapped to the sequence and the crystal structure of GlyRS (PDB 2PME).

Supplementary Figure 4 GlyRS stabilizes Ubc12N8.

Time courses of Ubc12N8 degradation in the absence and presence of WT or Δ232-238 GlyRS. Gels were stained by coomassie blue and quantified with ImageJ.

Supplementary Figure 5 The ABD of GlyRS and the APPBP1 subunit of E1 are responsible for the GlyRS-E1 interaction.

(a) GST pull-down assay to detect the interaction of GST-APPBP1-UBA3 with GlyRS and its truncated fragments. (b) Molecular docking of GlyRS (PDB 2PME) and APPBP1-UBA3 (PDB 2NVU) by using Patchdock.

Supplementary Figure 6 GlyRS facilitates cullin neddylation.

(a) Modeling analysis suggesting that GlyRS is unlikely to interfere with NEDD8 transferring from Ubc12 to cullin. The complex structure of cullin1-Rbx1-Ubc12N8 is adapted from PDB 4P5O. (b) Biolayer interferometry analysis to detect the binding of cullin1-Rbx1 (5 μg/mL) or GlyRS (5 μg/mL) or both to the immobilized Ubc12N8. Black dotted lines indicate the calculated sum of the binding curves for cullin1-Rbx1 and for GlyRS, respectively. (c) In vitro neddylation assay to detect the activity of WT and Δ232-238 GlyRS in promoting cullin1 neddylation.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 986 kb)

Supplementary Data Set 1

Combined raw gels and blots (PDF 6568 kb)

Supplementary Data Set 2

Model of the GlyRS–Ubc12N8 complex (PDB 651 kb)

Supplementary Data Set 3

Model of the GlyRS–E1 complex (PDB 950 kb)

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Mo, Z., Zhang, Q., Liu, Z. et al. Neddylation requires glycyl-tRNA synthetase to protect activated E2. Nat Struct Mol Biol 23, 730–737 (2016). https://doi.org/10.1038/nsmb.3250

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