Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

Nature Chemical Biology volume 11, pages 847–854 (2015)Cite this article

Subjects

Abstract

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The mouse XXYLT1 is a dimer and has a GT-A fold with its active site facing sideways to facilitate lateral modification of Notch.
Figure 2: The Xyl-Glc-EGF acceptor substrate undergoes a large conformational change when bound to XXYLT1.
Figure 3: Snapshots of XXYLT1 along its retaining reaction pathway.
Figure 4: Proposed retaining mechanism of XXYLT1.
Figure 5: Notch signaling in certain cancers may be inhibited by alterations in XXYLT1.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Kopan, R. & Ilagan, M.X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216–233 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ntziachristos, P., Lim, J.S., Sage, J. & Aifantis, I. From fly wings to targeted cancer therapies: a centennial for notch signaling. Cancer Cell 25, 318–334 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Takeuchi, H. & Haltiwanger, R.S. Significance of glycosylation in Notch signaling. Biochem. Biophys. Res. Commun. 453, 235–242 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Acar, M. et al. Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132, 247–258 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fernandez-Valdivia, R. et al. Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138, 1925–1934 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Takeuchi, H. et al. Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase. Proc. Natl. Acad. Sci. USA 108, 16600–16605 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sethi, M.K. et al. Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated Notch epidermal growth factor repeats. J. Biol. Chem. 285, 1582–1586 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Sethi, M.K. et al. Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of Notch. J. Biol. Chem. 287, 2739–2748 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Lee, T.V. et al. Negative regulation of Notch signaling by xylose. PLoS Genet. 9, e1003547 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Lairson, L.L., Henrissat, B., Davies, G.J. & Withers, S.G. Glycosyltransferases: structures, functions and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Breton, C., Fournel-Gigleux, S. & Palcic, M.M. Recent structures, evolution and mechanisms of glycosyltransferases. Curr. Opin. Struct. Biol. 22, 540–549 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Hurtado-Guerrero, R. & Davies, G.J. Recent structural and mechanistic insights into post-translational enzymatic glycosylation. Curr. Opin. Chem. Biol. 16, 479–487 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Chaikuad, A. et al. Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis. Proc. Natl. Acad. Sci. USA 108, 21028–21033 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, S.S. et al. Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase. Nat. Chem. Biol. 7, 631–638 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Lira-Navarrete, E. et al. Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase 2. Angew. Chem. Int. Ed. Engl. 53, 8206–8210 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Persson, K. et al. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol. 8, 166–175 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Errey, J.C. et al. Mechanistic insight into enzymatic glycosyl transfer with retention of configuration through analysis of glycomimetic inhibitors. Angew. Chem. Int. Ed. Engl. 49, 1234–1237 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Takeuchi, H., Kantharia, J., Sethi, M.K., Bakker, H. & Haltiwanger, R.S. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of Notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J. Biol. Chem. 287, 33934–33944 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Taylor, P. et al. Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands. Proc. Natl. Acad. Sci. USA 111, 7290–7295 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Minamida, S. et al. Detection of UDP-d-xylose: alpha-d-xyloside α1→3xylosyltransferase activity in human hepatoma cell line HepG2. J. Biochem. 120, 1002–1006 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Wiggins, C.A. & Munro, S. Activity of the yeast MNN1 α-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl. Acad. Sci. USA 95, 7945–7950 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wouters, M.A. et al. Evolution of distinct EGF domains with specific functions. Protein Sci. 14, 1091–1103 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Rao, Z. et al. The structure of a Ca2+-binding epidermal growth factor–like domain: its role in protein-protein interactions. Cell 82, 131–141 (1995).

    Article  CAS  PubMed  Google Scholar 

  25. Cordle, J. et al. A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nat. Struct. Mol. Biol. 15, 849–857 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kitamura, M., Hojo, H., Nakahara, Y., Ishimizu, T. & Hase, S. Synthesis and conformational characterization of the epidermal growth factor–like domain of blood coagulation factor IX carrying xylosyl-glucose. Glycoconj. J. 21, 197–203 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Nishimura, H., Takao, T., Hase, S., Shimonishi, Y. & Iwanaga, S. Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue. J. Biol. Chem. 267, 17520–17525 (1992).

    CAS  PubMed  Google Scholar 

  28. Lairson, L.L. et al. Intermediate trapping on a mutant retaining α-galactosyltransferase identifies an unexpected aspartate residue. J. Biol. Chem. 279, 28339–28344 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Schuman, B., Evans, S.V. & Fyles, T.M. Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism. PLoS ONE 8, e71077 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gómez, H., Polyak, I., Thiel, W., Lluch, J.M. & Masgrau, L. Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion–like transition state. J. Am. Chem. Soc. 134, 4743–4752 (2012).

    Article  PubMed  Google Scholar 

  31. Klinakis, A. et al. A novel tumor suppressor function for the Notch pathway in myeloid leukemia. Nature 473, 230–233 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, N.J. et al. Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proc. Natl. Acad. Sci. USA 108, 17761–17766 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Agrawal, N. et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science 333, 1154–1157 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    Article  PubMed  Google Scholar 

  35. Ardèvol, A. & Rovira, C. The molecular mechanism of enzymatic glycosyl transfer with retention of configuration: evidence for a short-lived oxocarbenium-like species. Angew. Chem. Int. Ed. Engl. 50, 10897–10901 (2011).

    Article  PubMed  Google Scholar 

  36. Jung, K.H. & Schmidt, R.R. in Carbohydrate-Based Drug Discovery (ed. Wong, C.-H.) 609–659 (Wiley-VCH, 2003).

  37. Gloster, T.M. & Vocadlo, D.J. Developing inhibitors of glycan processing enzymes as tools for enabling glycobiology. Nat. Chem. Biol. 8, 683–694 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  39. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Pannu, N.S., Murshudov, G.N., Dodson, E.J. & Read, R.J. Incorporation of prior phase information strengthens maximum-likelihood structure refinement. Acta Crystallogr. D Biol. Crystallogr. 54, 1285–1294 (1998).

    Article  CAS  PubMed  Google Scholar 

  43. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schrodinger, L.L.C. The PyMOL Molecular Graphics System, Version 1.3r1 (2010).

  45. Pettersen, E.F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Vivian, J.T. & Callis, P.R. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys. J. 80, 2093–2109 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bräm, O. et al. Relaxation dynamics of tryptophan in water: A UV fluorescence up-conversion and molecular dynamics study. J. Phys. Chem. A 114, 9034–9042 (2010).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank members of the Li and Haltiwanger labs for critical comments on this work. The work was supported by US National Institutes of Health grants GM061126 (R.S.H.) and AG029979 (H.L.), Stony Brook University–Brookhaven National Laboratory Seed grant (R.S.H. and H.L.), German Research Foundation (Deutsche Forschungsgemeinschaft (DFG)) grant BA4091/5-1 (H.B.) and National Science Foundation grant DMR 1404985 (E.L.). We acknowledge access to beamlines X25, X29 and X6A at the National Synchrotron Light Source (NSLS) and thank the staff at these beamlines. NSLS were supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-98CH10886. UDP-xylose isolation by the Carbosource Services at the Complex Carbohydrate Research Center, University of Georgia was supported in part by the US Department of Energy grant DE-FG02-93ER20097. The results published here are in part based upon data generated by the Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/).

Author information

Author notes

  1. Megumi Takeuchi, Robert S Haltiwanger & Hideyuki Takeuchi

    Present address: Present address: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.,

Authors and Affiliations

  1. Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA

    Hongjun Yu & Huilin Li

  2. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Megumi Takeuchi, Joshua Kantharia, Erwin London, Robert S Haltiwanger, Huilin Li & Hideyuki Takeuchi

  3. Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA

    Jamie LeBarron

  4. Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany

    Hans Bakker

Authors
  1. Hongjun Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Megumi Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jamie LeBarron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joshua Kantharia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Erwin London
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans Bakker
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert S Haltiwanger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Huilin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hideyuki Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.Y., H.B., R.S.H., H.L. and H.T. designed the research. H.Y. prepared the complexes, performed crystallization and solved the structures. H.Y. and H.L. analyzed the structures. M.T. and H.T. expressed and purified proteins and mutants, and performed the enzymatic assays. H.Y., R.S.H., H.L. and H.T. designed the mutants, analyzed the mutant data, performed the cancer-related analysis and wrote the paper. J.L., J.K. and E.L. joined this project after the original submission of the manuscript and contributed to the biophysical experiments regarding the folding status of wild-type and mutated XXYLT1 proteins, which was essential for the revisions to the manuscript.

Corresponding authors

Correspondence to Huilin Li or Hideyuki Takeuchi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–4. (PDF 9008 kb)

A movie morphing the isolated hFA9 EGF (PDB 1EDM) to the same protein in complex with XXYLT1 (MOV 19050 kb)

41589_2015_BFnchembio1927_MOESM292_ESM.mov

Morphing (generated using Chimera) of the electron densities of donor and acceptor substrates of the four trapped ternary complexes. (MOV 13771 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, H., Takeuchi, M., LeBarron, J. et al. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Nat Chem Biol 11, 847–854 (2015). https://doi.org/10.1038/nchembio.1927

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1927

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer