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Structural basis of tubulin detyrosination by the vasohibin–SVBP enzyme complex

Abstract

Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.

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Data availability

Coordinates of the X-ray crystal structures have been deposited in the RCSB PDB (www.rcsb.org) with the following accession numbers: V2c–SVBP (6J4O and 6QBY), V2c–SVBP–epoY (6J4P), V2c–SVBP–TPCK (6J4Q), V2c–SVBP–mαTail (6J4V), V2–SVBP (6J4S) and V1c–SVBP (6J4U). Uncropped blot/gel images are shown in Supplementary Data Set 1. Source data for Figs. 4h,i, 5d–h and 6a–c, Supplementary Fig. 2, Supplementary Fig. 4 and Supplementary Fig. 5f are available in Supplementary Data Set 2. Other data are available upon reasonable request.

References

  1. 1.

    Barisic, M. et al. Microtubule detyrosination guides chromosomes during mitosis. Science 348, 799–803 (2015).

  2. 2.

    Robison, P. et al. Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes. Science 352, aaf0659 (2016).

  3. 3.

    Chen, C. Y. et al. Suppression of detyrosinated microtubules improves cardiomyocyte function in human heart failure. Nat. Med. 24, 1225–1233 (2018).

  4. 4.

    Gobrecht, P. et al. Promotion of functional nerve regeneration by inhibition of microtubule detyrosination. J. Neurosci. 36, 3890–3902 (2016).

  5. 5.

    Marcos, S. et al. Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone. PLoS ONE 4, e5405 (2009).

  6. 6.

    Kerr, J. P. et al. Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle. Nat. Commun. 6, 8526 (2015).

  7. 7.

    Badin-Larcon, A. C. et al. Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin. Proc. Natl Acad. Sci. USA 101, 5577–5582 (2004).

  8. 8.

    Konishi, Y. & Setou, M. Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Nat. Neurosci. 12, 559–567 (2009).

  9. 9.

    Peris, L. et al. Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. J. Cell Biol. 174, 839–849 (2006).

  10. 10.

    Tas, R. P. et al. Differentiation between oppositely oriented microtubules controls polarized neuronal transport. Neuron 96, 1264–1271 (2017).

  11. 11.

    Gundersen, G. G. & Bulinski, J. C. Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis. J. Cell Biol. 102, 1118–1126 (1986).

  12. 12.

    Erck, C. et al. A vital role of tubulin-tyrosine-ligase for neuronal organization. Proc. Natl Acad. Sci. USA 102, 7853–7858 (2005).

  13. 13.

    Lafanechere, L. et al. Suppression of tubulin tyrosine ligase during tumor growth. J. Cell Sci. 111, 171–181 (1998).

  14. 14.

    Whipple, R. A. et al. Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement. Cancer Res. 70, 8127–8137 (2010).

  15. 15.

    Belmadani, S., Pous, C., Ventura-Clapier, R., Fischmeister, R. & Mery, P. F. Post-translational modifications of cardiac tubulin during chronic heart failure in the rat. Mol. Cell Biochem. 237, 39–46 (2002).

  16. 16.

    Magiera, M. M., Singh, P., Gadadhar, S. & Janke, C. Tubulin posttranslational modifications and emerging links to human disease. Cell 173, 1323–1327 (2018).

  17. 17.

    Westermann, S. & Weber, K. Post-translational modifications regulate microtubule function. Nat. Rev. Mol. Cell Biol. 4, 938–947 (2003).

  18. 18.

    Nieuwenhuis, J. & Brummelkamp, T. R. The tubulin detyrosination cycle: function and enzymes. Trends Cell Biol. 29, 80–92 (2019).

  19. 19.

    Murofushi, H. Purification and characterization of tubulin-tyrosine ligase from porcine brain. J. Biochem. 87, 979–984 (1980).

  20. 20.

    Ersfeld, K. et al. Characterization of the tubulin-tyrosine ligase. J. Cell Biol. 120, 725–732 (1993).

  21. 21.

    Prota, A. E. et al. Structural basis of tubulin tyrosination by tubulin tyrosine ligase. J. Cell Biol. 200, 259–270 (2013).

  22. 22.

    Szyk, A., Deaconescu, A. M., Piszczek, G. & Roll-Mecak, A. Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin. Nat. Struct. Mol. Biol. 18, 1250–1258 (2011).

  23. 23.

    Nieuwenhuis, J. et al. Vasohibins encode tubulin detyrosinating activity. Science 358, 1453–1456 (2017).

  24. 24.

    Aillaud, C. et al. Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science 358, 1448–1453 (2017).

  25. 25.

    Bieling, P. et al. CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233 (2008).

  26. 26.

    Peris, L. et al. Motor-dependent microtubule disassembly driven by tubulin tyrosination. J. Cell Biol. 185, 1159–1166 (2009).

  27. 27.

    Kreitzer, G., Liao, G. & Gundersen, G. G. Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol. Biol. Cell 10, 1105–1118 (1999).

  28. 28.

    Dunn, S. et al. Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells. J. Cell Sci. 121, 1085–1095 (2008).

  29. 29.

    Steinmetz, M. O. & Akhmanova, A. Capturing protein tails by CAP-Gly domains. Trends Biochem. Sci. 33, 535–545 (2008).

  30. 30.

    Sanchez-Pulido, L. & Ponting, C. P. Vasohibins: new transglutaminase-like cysteine proteases possessing a non-canonical Cys–His–Ser catalytic triad. Bioinformatics 32, 1441–1445 (2016).

  31. 31.

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

  32. 32.

    Chatterjee, D., Boyd, C. D., O’Toole, G. A. & Sondermann, H. Structural characterization of a conserved, calcium-dependent periplasmic protease from Legionella pneumophila. J. Bacteriol. 194, 4415–4425 (2012).

  33. 33.

    Stieler, M. et al. Structure of active coagulation factor XIII triggered by calcium binding: basis for the design of next-generation anticoagulants. Angew. Chem. Int. Ed. Engl. 52, 11930–11934 (2013).

  34. 34.

    Xu, X. et al. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes: structure of Mesorhizobium loti arylamine N-acetyltransferase in complex with coenzyme A. Acta Crystallogr. D. Biol. Crystallogr. 71, 266–273 (2015).

  35. 35.

    Auld, D. S. in Handbook of Proteolytic Enzymes 3rd edn (eds Rawlings, N. D. & Alvesen, G.) 1289–1301 (Academic Press, 2013).

  36. 36.

    Watt, W. et al. The atomic-resolution structure of human caspase-8, a key activator of apoptosis. Structure 7, 1135–1143 (1999).

  37. 37.

    Kagawa, T. F. et al. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease. Proc. Natl Acad. Sci. USA 97, 2235–2240 (2000).

  38. 38.

    Suzuki, Y. et al. Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion. J. Cell Sci. 123, 3094–3101 (2010).

  39. 39.

    Saoudi, Y., Paintrand, I., Multigner, L. & Job, D. Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins. J. Cell Sci. 108, 357–367 (1995).

  40. 40.

    Sato, Y. The vasohibin family: a novel family for angiogenesis regulation. J. Biochem. 153, 5–11 (2013).

  41. 41.

    Aillaud, C. et al. Evidence for new C-terminally truncated variants of alpha- and beta-tubulins. Mol. Biol. Cell 27, 640–653 (2016).

  42. 42.

    Sonoda, H. et al. Multiple processing forms and their biological activities of a novel angiogenesis inhibitor vasohibin. Biochem. Biophys. Res. Commun. 342, 640–646 (2006).

  43. 43.

    Hornbeck, P. V. et al. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43, D512–D520 (2015).

  44. 44.

    Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010).

  45. 45.

    Skubak, P. & Pannu, N. S. Automatic protein structure solution from weak X-ray data. Nat. Commun. 4, 2777 (2013).

  46. 46.

    Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution-from diffraction images to an initial model in minutes. Acta Crystallogr. D 62, 859–866 (2006).

  47. 47.

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

  48. 48.

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

  49. 49.

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

  50. 50.

    Cowtan, K. Completion of autobuilt protein models using a database of protein fragments. Acta Crystallogr. D 68, 328–335 (2012).

  51. 51.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  52. 52.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  53. 53.

    Lieuvin, A., Labbé, J. C., Dorée, M. & Job, D. Intrinsic microtubule stability in interphase cells. J. Cell Biol. 124, 985–996 (1994).

  54. 54.

    Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

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Acknowledgements

We thank N. E. Duggan, F. Vossier and L. Demacedo for technical assistance and the zootechnicians of the Grenoble Institute Neuroscience (GIN) for animal care. We thank J. Brocard for valuable discussions on data quantification. X-ray data were collected at beamlines BL-18U/19U of the Shanghai Synchrotron Radiation Facility (Shanghai, China) and X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland). This work was supported by grants from the National Key R&D Program of China (grant no. 2018YFC1004500), PIC-GIN (Photonic Imaging Center of GIN, University Grenoble Alpes-Inserm U1216, which is part of the ISdV core facility and certified by the IBiSA label), INSERM, CEA, University Grenoble Alpes, CNRS, La Ligue Contre le Cancer comité de l’Isère (to M.-J.M.), fondation France Alzheimer (AAP Sciences Médicales 2018, to M.-J.M.), the Swiss National Science Foundation (grant no. 31003A_166608; to M.O.S.), the Shenzhen Government (‘Peacock Plan’ No. Y01226136; to H.H.) and the Thousand Young Talents Program (to H.H.).

Author information

N.W. designed and performed cloning experiments, prepared recombinant proteins, crystallized V2c–SVBP, V2c–SVBP–epoY, V2c–SVBP–TPCK, V2c–SVBP–mαTail, V2–SVBP and V1c–SVBP complexes, performed GST pulldown experiments and analyzed the structures. C.B. analyzed sequences, and designed and performed cloning experiments for expression in mammalian cells. S.R.C. analyzed sequences, designed and performed cloning experiments, prepared recombinant proteins, performed microtubule-binding experiments, crystallized V2c–SVBP complex and performed biophysical and biochemical experiments. B.B. performed cycling cell-based experiments and statistical analyses. L.P. performed neuronal experiments and statistical analyses. N.O. analyzed sequences, designed and performed cloning experiments and prepared proteins. H.B. collected and processed X-ray data on V2c–SVBP, V2c–SVBP–epoY, V2c–SVBP–TPCK, V2c–SVBP–mαTail, V2–SVBP and V1c–SVBP crystals. V.O. collected and processed X-ray data and solved the second V2c–SVBP structure. F.K. performed the in vitro detyrosination assays. L.C. prepared some recombinant proteins. A.A. provided intellectual input to the project. M.-J.M. performed enzymatic activity measurements, supervised the functional experiments with mammalian cells, analyzed all the data and wrote the paper with input from all authors. M.O.S. and H.H. supervised the crystallography and biochemistry experiments, analyzed all the data and wrote the paper with input from all authors.

Correspondence to Marie-Jo Moutin or Michel O. Steinmetz or Hongda Huang.

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Integrated supplementary information

Supplementary Figure 1 Comparison of different crystal structures of vasohibin–SVBP.

a, Superimposition of the structures of V2c–SVBP (colored as in Fig. 1a) and V2–SVBP (colored in wheat) in cartoon representation. The rmsd of the two superimposed structures is 0.29 Å. The comparison reveals that the structures of V2c and full-length V2 are almost the same, and that the N- and C-terminal flanking regions of V2 are disordered. b, Superimposition of the structures of V2c–SVBP (colored as in Fig. 1a) and V1c–SVBP (colored in wheat) in cartoon representation. Though full-length SVBP was used for all the structure determinations, it should be noted that the α-helix of SVBP observed in the V1c–SVBP structure is two turns shorter as compared to the one observed in the V2c–SVBP structure. The rmsd of the two superimposed structures is 0.80 Å. The comparison reveals that the structures of V2c and V1c are very similar. c, Immunoblot of tubulin detyrosination assays performed in HEK293T cells with different V2 variants, including wild-type V2 and V2c and active site V2 mutants. Plasmids encoding wild type or mutated V2-eGFP and wild type SVBP-myc-Flag were cotransfected into cells. Antibodies specific to detyrosinated tubulin are used to assess detyrosinase activity. Antibodies against GAPDH, eGFP (V2 and variants), and Flag (SVBP) reveal the amounts of protein in the extract. Non-transfected cells reveal the endogenous levels of detyrosinated tubulin. (+) refers to wild type V2 or SVBP proteins. Immunoblots are representative of results obtained from at least three independent experiments (see Supplementary Fig. 2a). Uncropped images are shown in Supplementary Data Set 1.

Supplementary Figure 2 Quantification of tubulin detyrosination assay immunoblots.

Quantification results of immunoblots corresponding to Supplementary Fig. 1c (a) and Figs 2c (b), 2f (c), 3c (d), 4f (e), 4g (f), and 5b (g). Immunoblots, resulting each from an independent transfection, were analyzed for the negative control (sham transfection, white column, n = 16 total), the wild type non-mutated vasohibins (black columns, n = 3–5 per graph) or mutated vasohibins (gray columns, n = 3 except for F186A and Y236A in panel a (n = 2), and W63E W67E in panel e (n = 4)), and expressed as mean ± sem. In the top graphs, the plotted values represent the relative intensities of the detyrosinated-tubulin (deTyr-tub) signal obtained with mutants after normalization to the GAPDH signal. The signal of the wild type vasohibin from the same blots was set to 1.0, upper dotted line. Note that this measurement corresponds to endogenous (bottom dotted line) plus exogenous detyrosination activities in cells. In the middle graphs, the plotted values represent the relative quantities of mutant vasohibins expressed in cells after normalization to the GAPDH signal. The signal of the wild type vasohibin from the same blots was set to 1.0, upper dotted line. Note that this signal only corresponds to exogenous vasohibin levels since detection was performed using an anti-eGFP antibody. In the bottom graphs, the plotted values represent the relative intensities of the deTyr-tubulin signal obtained with mutants after subtracting the HEK293T cells endogenous levels of deTyr-tubulin (obtained with sham transfected cells, white columns in top graphs). The signal obtained with wild type vasohibin from the same blots was set to 1.0, upper dotted line. All The source files are available in Supplementary Data Set 2.

Supplementary Figure 3 Characteristics of the positively charged groove of vasohibins and inhibition of vasohibins by small molecules.

a–b, 14 conserved lysine and arginine residues (see also Supplementary Note) shape the positively charged groove of the V2c–SVBP (a) and V1c–SVBP (b) structures. c, Chemical structure of epoY that inhibits vasohibins with very good potency. d, Superimposition of the structures of V2c–SVBP (color coded as in Fig. 1a) and V2c–SVBP–epoY (wheat) in cartoon representation with the epoY shown in sticks representation. The rmsd of the two superimposed structures is 0.61 Å. e, Related to (d), walleye stereo view highlighting the covalent linkage between epoY and the catalytic residue C158 of V2c. f, Superimposition of the active site residues of V2c-SVBP (green) onto the ones of V2c–SVBP–epoY (wheat) in sticks representation. The red arrows highlight the conformational changes that residues experience upon ligand binding. g, Chemical structure of TPCK that inhibits vasohibins with very good potency. h, Superimposition of the structures of V2c–SVBP (color coded as in Fig. 1a) and V2c–SVBP–TPCK (wheat) in cartoon representation with the ligand TPCK shown in sticks. The rmsd is 0.43 Å. i, Superimposition of the active site residues of V2c–SVBP (green) onto the ones of V2c–SVBP–TPCK (wheat) in sticks representation. The red arrows highlight the conformational changes that residues experience upon ligand binding. j–l, Chemical structures of TLCK (j), E-64 (k) and parthenolide (l). The ligands TLCK, E-64 and parthenolide only show modest inhibitory activity against vasohibins. m, Zoom in view on the active site residues of the structure of carboxypeptidase A (CPA) in complex with a Gly-Tyr dipeptide (PDB ID 3CPA). Interacting residues of CPA (green) with the Gly-Tyr dipeptide (wheat) are shown in sticks representation. Both the catalytic residue E270 and the catalytic zinc atom are also highlighted, with the zinc-coordinating residues omitted for clarity.

Supplementary Figure 4 Analysis of the initial rates of activity of purified catalytic site mutants.

a–j, In vitro detyrosination assays were performed with purified V2–SVBP (composed of wild type or mutated full length V2 and wild-type SVBP) at the concentrations indicated using [14C]-tyrosinated taxol-stabilized microtubules (2 μM). n = 2–4 data points per time point and V2–SVBP concentration. n.d., not determined. Linear regression lines are shown in black. All the source files are available in Supplementary Data Set 2. The correct folding of proteins was assessed by size exclusion chromatography (Supplementary Fig. 6b–h).

Supplementary Figure 5 Analysis of the V2c–SVBP–mαTail structure and characterization of the vasohibin–SVBP interaction.

a, Sequence alignment of the C-terminal tails of the human α-tubulin isotypes. Identical and conserved residues are highlighted. The sequence of the mαTail used for crystallization is derived from the TUBA-1A/B isotypes and shown below the alignment. The 447-EGECY-451 segment (colored in red) of the mαTail is resolved and modeled in the V2c–SVBP–mαTail structure, while the other residues of the mαTail are disordered in the structure. b, Superimposition of the structures of V2c–SVBP (color coded as in Fig. 1a) and V2c–SVBP–mαTail (wheat) in cartoon representations, with the mαTail peptide (wheat) shown in sticks representation. The rmsd of these two superimposed structures is 0.19 Å. c, Superimposition of the structures of V2c–SVBP–epoY (color coded as in Fig. 1a) and V2c–SVBP–mαTail (wheat) in cartoon representations, with the epoY (green) and mαTail peptide (wheat) shown in sticks representations. The rmsd of these two superimposed structures is 0.46 Å. d, Related to panel (c), walleye stereo view of the ligand epoY (green) and the peptide mαTail (wheat) after superimposition of the V2c–SVBP–epoY and V2c–SVBP–mαTail structures. e, Overall view of the interacting structural elements between V2c and SVBP in the V2c–SVBP structure in cartoon representation. The SVBP α-helix (magenta) is wrapped around by the N-terminal domain (ND; cyan) of V2c and is further contacted by the C-terminal domain (CD; green) of V2c through the three loops L4, L5 and L9 (highlighted with wheat tubules). Detailed interactions between V2c and SVBP are showed in Fig. 4a–c. f, ITC experiment with purified MBP-V2c and SVBP proteins. The source file is available in Supplementary Data Set 2. g, SDS-PAGE of GST pull-down experiments performed with different MBP-V2 mutants and wild type GST-SVBP, (see also Fig. 4d). Both distinct V2 double point mutations impair the interaction with SVBP. Uncropped images are shown in Supplementary Data Set 1.

Supplementary Figure 6 Size exclusion chromatography profiles of mutants.

a, Size exclusion chromatography profile of V2c 4A mutant. The V2c 4A–SVBP complex was compared with the wild type V2c–SVBP complex using size exclusion chromatography. The profile suggests that the V2 4A mutant is indeed folded properly. b–h, Most of the complex mutants used in this study were also analyzed and compared with the wild type V2–SVBP complexes using size exclusion chromatography. The profiles also suggest that all V2 variants are folded properly.

Supplementary Figure 7 The vasohibin–SVBP interaction is crucial for axon specification.

a, Wild type human SVBP (hSVBP), but not its double mutant I39A Y40A, rescues axonal specification in mouse hippocampal neurons knocked down for mouse SVBP (mSVBP). Representative immunofluorescence images of neurons cotransfected with plasmids expressing a shRNA (CTRL or mSVBP) and hSVBP-myc-Flag (wild type or I39A Y40A). Tyrosinated and detyrosinated α-tubulin contents were assayed at 1DIV and staining images were merged. Detyrosinated tubulin levels and number of stage III neuron (bearing an axon) are significantly reduced in knocked down neurons (top right: sh mSVBP) compared to control neurons (top left: sh control), and rescued with native hSVBP (bottom left: sh mSVBP + hSVBP) but not with the hSVBP I39A Y40A mutant (bottom right: sh mSVBP + hSVBP I39A Y40A). Scale bar is indicated. Arrowheads and arrows highlight single and double axons emanating from a cell body, respectively.

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Supplementary Figs. 1–7, Supplementary Note 1

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Supplementary Data Set 1

Supplementary Data Set 2

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Further reading

Fig. 1: Structural analysis of the vasohibin–SVBP enzyme complex.
Fig. 2: Vasohibin–SVBP active site architecture and molecular basis of inhibition.
Fig. 3: Interaction of vasohibin–SVBP with a C-terminal α-tubulin peptide.
Fig. 4: Role of the vasohibin-SVBP interaction.
Fig. 5: Determinants mediating association of the V2–SVBP complex with microtubules.
Fig. 6: Role of vasohibin-SVBP-microtubule interaction in the regulation of axon specification.
Fig. 7: Structural basis of the tubulin detyrosination–tyrosination cycle.
Supplementary Figure 1: Comparison of different crystal structures of vasohibin–SVBP.
Supplementary Figure 2: Quantification of tubulin detyrosination assay immunoblots.
Supplementary Figure 3: Characteristics of the positively charged groove of vasohibins and inhibition of vasohibins by small molecules.
Supplementary Figure 4: Analysis of the initial rates of activity of purified catalytic site mutants.
Supplementary Figure 5: Analysis of the V2c–SVBP–mαTail structure and characterization of the vasohibin–SVBP interaction.
Supplementary Figure 6: Size exclusion chromatography profiles of mutants.
Supplementary Figure 7: The vasohibin–SVBP interaction is crucial for axon specification.