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.

  • Brief Communication
  • Published:

Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1–SVBP

Nature Structural & Molecular Biology volume 26, pages 567–570 (2019)Cite this article

Subjects

Abstract

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.

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

Fig. 1: X-ray structure of the human VASH1–SVBP complex.
Fig. 2: Structural analysis of the VASH1–SVBP complex.
Fig. 3: Tubulin tail binding and recognition.

Similar content being viewed by others

Data availability

The coordinates for the crystal structure of the VASH1–SVBP complex are deposited in the PDB under the accession identifier PDB 6NVQ. All other data and the coordinates of the HADDOCK model are available upon request.

References

  1. Wehenkel, A. & Janke, C. Nat. Cell Biol. 16, 303–305 (2014).

    Article  CAS  Google Scholar 

  2. Murofushi, H. J. Biochem. 87, 979–984 (1980).

    Article  CAS  Google Scholar 

  3. Prota, A. E. et al. J. Cell Biol. 200, 259–270 (2013).

    Article  CAS  Google Scholar 

  4. Szyk, A., Deaconescu, A. M., Piszczek, G. & Roll-Mecak, A. Nat. Struct. Mol. Biol. 18, 1250–1258 (2011).

    Article  CAS  Google Scholar 

  5. Nieuwenhuis, J. et al. Science 358, eaao5676–1456 (2017).

    Article  Google Scholar 

  6. Aillaud, C. et al. Science 358, 1448–1453 (2017).

    Article  CAS  Google Scholar 

  7. Sanchez-Pulido, L. & Ponting, C. P. Bioinformatics 32, 1441–1445 (2016).

    Article  CAS  Google Scholar 

  8. Waltersperger, S. et al. J. Synchrotron Radiat. 22, 895–900 (2015).

    Article  Google Scholar 

  9. Rambo, R. P. & Tainer, J. A. Biopolymers 95, 559–571 (2011).

    Article  CAS  Google Scholar 

  10. Petoukhov, M. V. & Svergun, D. I. Biophys J 89, 1237–1250 (2005).

    Article  CAS  Google Scholar 

  11. van Zundert, G. C. P. et al. J. Mol. Biol. 428, 720–725 (2016).

    Article  Google Scholar 

  12. Otto, H.-H. & Schirmeister, T. Chem. Rev. 97, 133–172 (1997).

    Article  CAS  Google Scholar 

  13. Zhu, M., Shao, F., Innes, R. W., Dixon, J. E. & Xu, Z. Proc. Natl Acad. Sci. USA 101, 302–307 (2004).

    Article  CAS  Google Scholar 

  14. Luna-Vargas, M. P. A. et al. J. Struct. Biol. 175, 113–119 (2011).

    Article  CAS  Google Scholar 

  15. Newman, J. et al. Acta Crystallogr. D Biol. Crystallogr. 61, 1426–1431 (2005).

    Article  Google Scholar 

  16. Nurizzo, D. et al. Acta Crystallogr. D Struct. Biol. 72, 966–975 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Pape, T. & Schneider, T. J. Synchr. Rad. 37, 843–844 (2004).

    CAS  Google Scholar 

  19. Uson, I. & Sheldrick, G. Curr. Opin. Struct. Biol. 9, 643–648 (1999).

    Article  CAS  Google Scholar 

  20. Ness, S., de Graaff, R., Abrahams, J. & Pannu, N. Struct. Solut. Struct. (Camb.) 12, 1753–1761 (2004).

    Article  CAS  Google Scholar 

  21. Murshudov, G. N. et al. Macromol. Cryst. Struct. 67, 355–367 (2011).

    CAS  Google Scholar 

  22. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  23. Joosten, R. P., Long, F., Murshudov, G. N. & Perrakis, A. IUCrJ 1, 213–220 (2014).

    Article  CAS  Google Scholar 

  24. Chen, V. B. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  25. Petoukhov, M. V. et al. J. Appl. Crystallogr. 45, 342–350 (2012).

    Article  CAS  Google Scholar 

  26. Svergun, D., Barberato, C. & Koch, M. H. J. J. Appl. Crystallogr. 28, 768–773 (1995).

    Article  CAS  Google Scholar 

  27. Dominguez, C., Boelens, R. & Bonvin, A. M. J. J. J. Am. Chem. Soc. 125, 1731–1737 (2003).

    Article  CAS  Google Scholar 

  28. Holm, L. & Laakso, L. M. Nucleic Acids Res. 44, W351–W355 (2016).

    Article  CAS  Google Scholar 

  29. Sievers, F. et al. Mol. Syst. Biol. 7, 539–539 (2011).

    Article  Google Scholar 

  30. Chojnacki, S., Cowley, A., Lee, J., Foix, A. & Lopez, R. Nucleic Acids Res. 45, W550–W553 (2017).

    Article  CAS  Google Scholar 

  31. Ashkenazy, H. et al. Nucleic Acids Res. 44, W344–W350 (2016).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank V. Olieric for help with S-SAD phasing and data collection at PXIII at the SLS Synchrotron, and the MASSIF beamline team at the ESRF for native data collection. The authors received the following funding support: grant no. NWO 184.032.201 to M.A.; grant nos. NWO 016.Vici.170.033 and KWF NKI-2015–7609, support by the Cancer Genomics Center (CGC.nl) and an Ammodo KNAW Award 2015 to T.R.B.; support by the Oncode Institute to T.R.B. and A.P.

Author information

Author notes

  1. These authors contributed equally: Athanassios Adamopoulos, Lisa Landskron.

Authors and Affiliations

  1. Division of Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands

    Athanassios Adamopoulos, Lisa Landskron, Tatjana Heidebrecht, Foteini Tsakou, Joppe Nieuwenhuis, Patrick H. N. Celie, Thijn R. Brummelkamp & Anastassis Perrakis

  2. Oncode Institute, Division of Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands

    Athanassios Adamopoulos, Lisa Landskron, Tatjana Heidebrecht, Joppe Nieuwenhuis, Thijn R. Brummelkamp & Anastassis Perrakis

  3. Proteomics Facility, the Netherlands Cancer Institute, Amsterdam, the Netherlands

    Onno B. Bleijerveld & Maarten Altelaar

  4. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands

    Maarten Altelaar

  5. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Thijn R. Brummelkamp

  6. Cancer Genomics Center, Amsterdam, the Netherlands

    Thijn R. Brummelkamp

Authors
  1. Athanassios Adamopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisa Landskron
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatjana Heidebrecht
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Foteini Tsakou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Onno B. Bleijerveld
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maarten Altelaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joppe Nieuwenhuis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Patrick H. N. Celie
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thijn R. Brummelkamp
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Anastassis Perrakis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.A. performed and analyzed biochemical and structural experiments, wrote a draft manuscript and prepared figures. L.L. performed and analyzed cell-based experiments and prepared figures. T.H. participated in protein purification, crystallization and data collection. F.T. performed expression tests and produced initial crystals, under supervision of A.A. and P.C. O.B.B. and M.A. analyzed the MS samples prepared by J.N. A.P. participated in crystallographic data analysis, wrote the final manuscript and supervised the project together with T.R.B.

Corresponding author

Correspondence to Anastassis Perrakis.

Ethics declarations

Competing interests

T.R.B. is co-founder and SAB member of Haplogen GmbH and co-founder and managing director of Scenic Biotech. All other authors declare no competing interests.

Additional information

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

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Conservation of VASH1 and SVBP sequence.

Multiple sequence alignment of orthologous proteins for VASH1 and SVBP. Structural elements are highlighted based on the obtained crystal structure for the VASH1–SVBP complex.

Supplementary Figure 2 Analysis of the VASH1-SVBP interface.

a. Different views of the interacting surface between VASH1 and SVBP; VASH1 is shown as a surface, while SVBP is shown as ribbons; colors are according to conservation of the respective residues based on multiple sequence alignment of orthologous VASH1 proteins, as in Figure 2b. Axes are shown to compare the different views. b. Details for the quantification of detyrosination activity of the SVBP mutants shown in Figure 1e; each individual experiment value is shown as a separate point for the two replicates, and the bar represents the average.

Supplementary Figure 3 Characterization experiments for the VASH1-SVBP structure and the mode of recognition of the tubulin C-terminal peptide.

a. Mutation of a hydrogen bond in the crystallographic dimer of the VASH1–SVBP dimerization interface does not affect detyrosynation activity; detyrosination levels of HeLa cell lysates transfected with vectors expressing Flag-tagged SVBP, VASH1 and mutants were determined using western blotting; this experiments has been performed twice. b. Determination of VASH1 substrate specificity for the α-tubulin peptide based on the number of peptide spectrum matches (PSM) for the intact and cleaved/detyrosinated version of single-residue mutant peptides. c. a representative model from Cluster 1 of the first round of HADDOCK modeling; this model proved wrong on the basis of the validation experiments but is shown here as it was used to guide the first round of validation experiments.

Supplementary Figure 4 Quantification of detyrosination activity of VASH1 mutants.

a-c. The graphs relate to the quantification of activities shown in Figure 3; the bars present the mean and the two circles show the values of the two independent biological replicate experiments. d, e. The Western blot and the quantification establish that a mutation in His80 that was noticed in two of our mutants shown with an asterisk in Figure 3g, does not affect activity compared to the wild type; in the same experiment we confirm again that the K258E and K276E mutants on this background, have no significant effect on activity.

Supplementary Figure 5 Structural analysis of the active site of structural similar proteins to VASH1-SVBP complex.

Ribbon models of structural similar proteins based on DALI server; active site residues for each hit is shown as stick representation in all cases the catalytic triad contains a cysteine and a histidine, whereas the third position is occupied by either a glutamate or an aspartate; PDB codes are: 4DMO for Bacillus cereus arylamine N-acetyltransferase 3 NAT3; 4FGP for LapG, 2PQT for human N-acetyltransferase-1 (the acetanilide molecule that has been covalently attached to the active site C69 is not shown in the figure); 1UKF for Avirulence protein from plant pathogen Pseudomonas syringae; 4RXV for RavJ protein fom Leugionella pneumophila.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Tables 1–4 and Supplementary Data Set 1

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Adamopoulos, A., Landskron, L., Heidebrecht, T. et al. Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1–SVBP. Nat Struct Mol Biol 26, 567–570 (2019). https://doi.org/10.1038/s41594-019-0254-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-019-0254-6

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