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:

A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

Nature Chemical Biology volume 12, pages 240–246 (2016)Cite this article

Subjects

Abstract

Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

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: Structure of CePOFUT2 in complex with GDP and HsTSR1.
Figure 2: Catalytic mechanism of POFUT2 and its preference of threonine over serine residues.
Figure 3: Interactions in the interface of CePOFUT2 in complex with GDP and HsTSR1.
Figure 4: Hydration structure and dynamics of CePOFUT2-GDP-fucose-HsTSR1 supports water-mediated binding.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 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  Google Scholar 

  2. Moremen, K.W., Tiemeyer, M. & Nairn, A.V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462 (2012).

    Article  CAS  Google Scholar 

  3. Luther, K.B. & Haltiwanger, R.S. Role of unusual O-glycans in intercellular signaling. Int. J. Biochem. Cell Biol. 41, 1011–1024 (2009).

    Article  CAS  Google Scholar 

  4. Sakaidani, Y. et al. O-linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions. Nat. Commun. 2, 583 (2011).

    Article  Google Scholar 

  5. 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  Google Scholar 

  6. Vasudevan, D. & Haltiwanger, R.S. Novel roles for O-linked glycans in protein folding. Glycoconj. J. 31, 417–426 (2014).

    Article  CAS  Google Scholar 

  7. Luca, V.C. et al. Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science 347, 847–853 (2015).

    Article  CAS  Google Scholar 

  8. Vasudevan, D., Takeuchi, H., Johar, S.S., Majerus, E. & Haltiwanger, R.S. Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism. Curr. Biol. 25, 286–295 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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  Google Scholar 

  11. Chen, C.I. et al. Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation. EMBO J. 31, 3183–3197 (2012).

    Article  CAS  Google Scholar 

  12. Lira-Navarrete, E. et al. Structural insights into the mechanism of protein O-fucosylation. PLoS One 6, e25365 (2011).

    Article  CAS  Google Scholar 

  13. Yan, C., Wu, F., Jernigan, R.L., Dobbs, D. & Honavar, V. Characterization of protein-protein interfaces. Protein J. 27, 59–70 (2008).

    Article  CAS  Google Scholar 

  14. Tan, K. et al. Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication. J. Cell Biol. 159, 373–382 (2002).

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Ricketts, L.M., Dlugosz, M., Luther, K.B., Haltiwanger, R.S. & Majerus, E.M. O-fucosylation is required for ADAMTS13 secretion. J. Biol. Chem. 282, 17014–17023 (2007).

    Article  CAS  Google Scholar 

  17. Wang, L.W. et al. O-fucosylation of thrombospondin type 1 repeats in ADAMTS-like-1/punctin-1 regulates secretion: implications for the ADAMTS superfamily. J. Biol. Chem. 282, 17024–17031 (2007).

    Article  CAS  Google Scholar 

  18. Lira-Navarrete, E. et al. Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase2. Angew. Chem. Int. Edn Engl. 53, 8206–8210 (2014).

    Article  CAS  Google Scholar 

  19. Gonzalez de Peredo, A. et al. C-mannosylation and O-fucosylation of thrombospondin type 1 repeats. Mol. Cell. Proteomics 1, 11–18 (2002).

    Article  CAS  Google Scholar 

  20. Sun, T., Lin, F.H., Campbell, R.L., Allingham, J.S. & Davies, P.L. An antifreeze protein folds with an interior network of more than 400 semi-clathrate waters. Science 343, 795–798 (2014).

    Article  CAS  Google Scholar 

  21. Parsegian, V.A., Rand, R.P. & Rau, D.C. Macromolecules and water: probing with osmotic stress. Methods Enzymol. 259, 43–94 (1995).

    Article  CAS  Google Scholar 

  22. Rajapaksha, A., Stanley, C.B. & Todd, B.A. Effects of macromolecular crowding on the structure of a protein complex: a small-angle scattering study of superoxide dismutase. Biophys. J. 108, 967–974 (2015).

    Article  CAS  Google Scholar 

  23. Naidoo, K.J. & Kuttel, M. Water structure about the dimer and hexamer repeat units of amylose from molecular dynamics computer simulations. J. Comput. Chem. 22, 445–456 (2001).

    Article  CAS  Google Scholar 

  24. Soper, A.K. The radial distribution functions of water and ice from 220 to 673 K and at pressures up to 400 MPa. Chem. Phys. 258, 121–137 (2000).

    Article  CAS  Google Scholar 

  25. Andersson, C. & Engelsen, S.B. The mean hydration of carbohydrates as studied by normalized two-dimensional radial pair distributions. J. Mol. Graph. Model. 17, 101–105, 131–133 (1999).

    Article  CAS  Google Scholar 

  26. Ball, P. Water as an active constituent in cell biology. Chem. Rev. 108, 74–108 (2008).

    Article  CAS  Google Scholar 

  27. Sahún-Roncero, M. et al. The mechanism of allosteric coupling in choline kinase α1 revealed by the action of a rationally designed inhibitor. Angew. Chem. Int. Edn Engl. 52, 4582–4586 (2013).

    Article  Google Scholar 

  28. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  31. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  32. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  33. Luo, Y., Nita-Lazar, A. & Haltiwanger, R.S. Two distinct pathways for O-fucosylation of epidermal growth factor-like or thrombospondin type 1 repeats. J. Biol. Chem. 281, 9385–9392 (2006).

    Article  CAS  Google Scholar 

  34. Lira-Navarrete, E. et al. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat. Commun. 6, 6937 (2015).

    Article  CAS  Google Scholar 

  35. Horcas, I. et al. WSXM: a software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 78, 013705 (2007).

    Article  CAS  Google Scholar 

  36. Lostao, A., Peleato, M.L., Gómez-Moreno, C. & Fillat, M.F. Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: direct visualization by in situ atomic force microscopy under different redox conditions. Biochim. Biophys. Acta 1804, 1723–1729 (2010).

    Article  CAS  Google Scholar 

  37. Marcuello, C., Arilla-Luna, S., Medina, M. & Lostao, A. Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level. Biochim. Biophys. Acta 1834, 665–676 (2013).

    Article  CAS  Google Scholar 

  38. Leonhard-Melief, C. & Haltiwanger, R.S. O-fucosylation of thrombospondin type 1 repeats. Methods Enzymol. 480, 401–416 (2010).

    Article  CAS  Google Scholar 

  39. Al-Shareffi, E. et al. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2. Glycobiology 23, 188–198 (2013).

    Article  CAS  Google Scholar 

  40. Case, D.A. et al. AMBER 14. (University of California, San Francisco, 2014).

  41. Kirschner, K.N. et al. GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J. Comput. Chem. 29, 622–655 (2008).

    Article  CAS  Google Scholar 

  42. Bayly, C.I., Cieplak, P., Cornell, W. & Kollman, P.A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  43. Frisch, M.J. et al. Gaussian 09, Revision D.01. (Gaussian, Inc., Wallingford, CT, 2009).

  44. Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W. & Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  45. Hornak, V. et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, 712–725 (2006).

    Article  CAS  Google Scholar 

  46. Andrea, T.A., Swope, W.C. & Andersen, H.C. The role of long ranged forces in determining the structure and properties of liquid water. J. Chem. Phys. 79, 4576–4584 (1983).

    Article  CAS  Google Scholar 

  47. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S.B. Engelsen (University of Copenhagen) for providing the software to calculate 2D-RDF functions and residence times for water molecules. We thank synchrotron radiation sources DLS (Oxford) and beamline I02 (experiment number MX10121-2), and Institute for Biocomputation and Physics of Complex Systems (BIFI), (Memento cluster) for supercomputer support. This work was supported by Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Ministerio de Economía y Competitividad (MEC; BFU2010-19504 to R.H.-G., CTQ2013-44367-C2-2-P to R.H.-G., CTQ2012-36365 to F.C.), the US National Institutes of Health (GM061126 and CA123071, both to R.S.H.), the Ramón y Cajal Programme (fellowship to G.J.-O.), Diputación General de Aragón (DGA; B89 to R.H.-G.) and the EU Seventh Framework Programme (2007–2013) under BioStruct-X (grant agreement 283570 and BIOSTRUCTX 5186, to R.H.-G.).

Author information

Author notes

  1. Hideyuki Takeuchi & Robert S Haltiwanger

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

  2. Jessika Valero-González and Christina Leonhard-Melief: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Zaragoza, Spain

    Jessika Valero-González, Erandi Lira-Navarrete, Gonzalo Jiménez-Osés, Cristina Hernández-Ruiz & Ramon Hurtado-Guerrero

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

    Christina Leonhard-Melief, Deepika Vasudevan, Hideyuki Takeuchi & Robert S Haltiwanger

  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA

    Gonzalo Jiménez-Osés

  4. Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, Logroño, Spain

    Gonzalo Jiménez-Osés & Francisco Corzana

  5. Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain

    María Carmen Pallarés & Anabel Lostao

  6. Estación Experimental de Aula Dei (EEAD-CSIC), Zaragoza, Spain

    Inmaculada Yruela

  7. Fundación Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Zaragoza, Spain

    Anabel Lostao & Ramon Hurtado-Guerrero

  8. Instituto de Investigaciones Sanitarias de Aragón (IIS-A), Zaragoza, Spain

    Ramon Hurtado-Guerrero

Authors
  1. Jessika Valero-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christina Leonhard-Melief
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erandi Lira-Navarrete
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gonzalo Jiménez-Osés
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cristina Hernández-Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. María Carmen Pallarés
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Inmaculada Yruela
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Deepika Vasudevan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Anabel Lostao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Francisco Corzana
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hideyuki Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Robert S Haltiwanger
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ramon Hurtado-Guerrero
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.H.-G. designed the crystallization construct and solved the crystal structure. J.V.-G., E.L.-N., C.H.-R. and R.H.-G. cloned the different constructs, purified the enzymes and crystallized the complex. R.H.-G. solved and refined the crystal structure. R.H.-G. and J.V.-G. performed the ITC experiments. G.J.-O. and F.C. performed the molecular dynamics experiments. C.L.-M., D.V., H.T. and R.S.H. cloned the different constructs for expression in mammalian cells and performed site-directed mutagenesis, analysis of enzymatic studies (including the mutants in this work), study of the nonprocessivity of CePOFUT2 and studies in mammalian cells (both the secretion and the activity experiments). M.C.P. and A.L. performed the AFM studies. I.Y. and R.H.-G. performed the multiple alignment of the TSRs and POFUT2s. R.H.-G. wrote the article with contributions from H.T., R.S.H., F.C., A.L. and G.J.-O. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ramon Hurtado-Guerrero.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–19. (PDF 14555 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Valero-González, J., Leonhard-Melief, C., Lira-Navarrete, E. et al. A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat Chem Biol 12, 240–246 (2016). https://doi.org/10.1038/nchembio.2019

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2019

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing