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.

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes

An Author Correction to this article was published on 13 August 2020

This article has been updated

Abstract

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Glutamylation has two general phases: initiation and elongation.
Fig. 2: TTLL6 preferentially elongates branched glutamates in α-tubulin C-terminal tails.
Fig. 3: NMR spectroscopy indicates that enzymatic 15N-13C-labeled glutamate addition occurs via an α-linkage type.
Fig. 4: Structures of TTLL6 in complex with ATP and the α-elongation tetrahedral intermediate analog.
Fig. 5: Structural basis for TTLL6 α-linked glutamate chain elongation activity.
Fig. 6: TTLL4 is a β-tubulin-specific glutamate chain initiase.
Fig. 7: Structure-based engineering of TTLL6.
Fig. 8: Structure of engineered TTLL6 reveals basis for elongation versus initiation activity.

Data availability

Atomic models and structure factors have been deposited at the wwProtein Data Bank under the following accession codes: wild-type TTLL6 complexed with ATP (PDB 6VZT), α-elongation analog (PDB 6VZU), initiation analog (PDB 6VZW), γ-elongation analog (PDB 6VZV), TTLL6 mutant in complex with α-elongation analog (PDB 6VZQ), initiation analog (PDB 6VZR) and γ-elongation analog (PDB 6VZS). Source data are provided with this paper.

Change history

References

  1. 1.

    Yu, I., Garnham, C. P. & Roll-Mecak, A. Writing and reading the tubulin code. J. Biol. Chem. 290, 17163–17172 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Redeker, V., Le Caer, J. P., Rossier, J. & Prome, J. C. Structure of the polyglutamyl side chain posttranslationally added to α-tubulin. J. Biol. Chem. 266, 23461–23466 (1991).

    CAS  PubMed  Google Scholar 

  3. 3.

    Wolff, A., Houdayer, M., Chillet, D., de Nechaud, B. & Denoulet, P. Structure of the polyglutamyl chain of tubulin: occurrence of alpha and gamma linkages between glutamyl units revealed by monoreactive polyclonal antibodies. Biol. Cell 81, 11–16 (1994).

    CAS  PubMed  Google Scholar 

  4. 4.

    van Dijk, J. et al. A targeted multienzyme mechanism for selective microtubule polyglutamylation. Mol. Cell 26, 437–448 (2007).

    PubMed  Google Scholar 

  5. 5.

    Janke, C. et al. Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308, 1758–1762 (2005).

    CAS  PubMed  Google Scholar 

  6. 6.

    Garnham, C. P. et al. Multivalent microtubule recognition by tubulin tyrosine ligase-like family glutamylases. Cell 161, 1112–1123 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Mukai, M. et al. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway. Biochemistry 48, 1084–1093 (2009).

    CAS  PubMed  Google Scholar 

  8. 8.

    Roll-Mecak, A. How cells exploit tubulin diversity to build functional cellular microtubule mosaics. Curr. Opin. Cell Biol. 56, 102–108 (2019).

    CAS  PubMed  Google Scholar 

  9. 9.

    Redeker, V. Mass spectrometry analysis of C-terminal posttranslational modifications of tubulins. Methods Cell Biol. 95, 77–103 (2010).

    CAS  PubMed  Google Scholar 

  10. 10.

    Audebert, S. et al. Reversible polyglutamylation of α- and β-tubulin and microtubule dynamics in mouse brain neurons. Mol. Biol. Cell 4, 615–626 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Janke, C. & Kneussel, M. Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci. 33, 362–372 (2010).

    CAS  PubMed  Google Scholar 

  12. 12.

    Schneider, A., Plessmann, U., Felleisen, R. & Weber, K. Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites. FEBS Lett. 429, 399–402 (1998).

    CAS  PubMed  Google Scholar 

  13. 13.

    Geimer, S., Teltenkotter, A., Plessmann, U., Weber, K. & Lechtreck, K. F. Purification and characterization of basal apparatuses from a flagellate green alga. Cell Motil. Cytoskeleton 37, 72–85 (1997).

    CAS  PubMed  Google Scholar 

  14. 14.

    Sirajuddin, M., Rice, L. M. & Vale, R. D. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat. Cell Biol. 16, 335–344 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lacroix, B. et al. Tubulin polyglutamylation stimulates spastin-mediated microtubule severing. J. Cell Biol. 189, 945–954 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Valenstein, M. L. & Roll-Mecak, A. Graded control of microtubule severing by tubulin glutamylation. Cell 164, 911–921 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Magiera, M. M. et al. Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J. 37, e100440 (2018).

  18. 18.

    Bodakuntla, S. et al. Tubulin polyglutamylation is a general traffic control mechanism in hippocampal neurons. J. Cell Sci. 133, jcs241802 (2020).

  19. 19.

    Suryavanshi, S. et al. Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity. Curr. Biol. 20, 435–440 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kubo, T., Yanagisawa, H. A., Yagi, T., Hirono, M. & Kamiya, R. Tubulin polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins. Curr. Biol. 20, 441–445 (2010).

    CAS  PubMed  Google Scholar 

  21. 21.

    Hong, S. R. et al. Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nat. Commun. 9, 1732 (2018).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

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

    CAS  PubMed  Google Scholar 

  23. 23.

    Zempel, H. et al. Amyloid-β oligomers induce synaptic damage via Tau-dependent microtubule severing by TTLL6 and spastin. EMBO J. 32, 2920–2937 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Lee, J. E. et al. CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium. Nat. Genet. 44, 193–199 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Xia, P. et al. Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity. Nat. Immunol. 17, 369–378 (2016).

    CAS  PubMed  Google Scholar 

  26. 26.

    Sun, X. et al. Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations. Proc. Natl Acad. Sci. USA 113, E2925–E2934 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Bedoni, N. et al. Mutations in the polyglutamylase gene TTLL5, expressed in photoreceptor cells and spermatozoa, are associated with cone-rod degeneration and reduced male fertility. Hum. Mol. Genet. 25, 4546–4555 (2016).

    CAS  PubMed  Google Scholar 

  28. 28.

    Dias, M. S. et al. Novel splice-site mutation in TTLL5 causes cone dystrophy in a consanguineous family. Mol. Vis. 23, 131–139 (2017).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Onikubo, T. et al. Developmentally regulated post-translational modification of nucleoplasmin controls histone sequestration and deposition. Cell Rep. 10, 1735–1748 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Miller, K. E. & Heald, R. Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus. J. Cell Biol. 209, 211–220 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ye, B. et al. Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice. Nat. Commun. 9, 1261 (2018).

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Kashiwaya, K. et al. Involvement of the tubulin tyrosine ligase-like family member 4 polyglutamylase in PELP1 polyglutamylation and chromatin remodeling in pancreatic cancer cells. Cancer Res. 70, 4024–4033 (2010).

    CAS  PubMed  Google Scholar 

  33. 33.

    Redeker, V., Rossier, J. & Frankfurter, A. Posttranslational modifications of the C-terminus of α-tubulin in adult rat brain: α4 is glutamylated at two residues. Biochemistry 37, 14838–14844 (1998).

    CAS  PubMed  Google Scholar 

  34. 34.

    Edde, B. et al. Posttranslational glutamylation of α-tubulin. Science 247, 83–85 (1990).

    CAS  PubMed  Google Scholar 

  35. 35.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Fan, C., Moews, P. C., Walsh, C. T. & Knox, J. R. Vancomycin resistance: structure of d-alanine:d-alanine ligase at 2.3 Å resolution. Science 266, 439–443 (1994).

    CAS  PubMed  Google Scholar 

  38. 38.

    Collinsova, M. & Jiracek, J. Phosphinic acid compounds in biochemistry, biology and medicine. Curr. Med. Chem. 7, 629–647 (2000).

    CAS  PubMed  Google Scholar 

  39. 39.

    Liu, Y., Garnham, C. P., Roll-Mecak, A. & Tanner, M. E. Phosphinic acid-based inhibitors of tubulin polyglutamylases. Bioorg. Med. Chem. Lett. 23, 4408–4412 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Duncan, K. & Walsh, C. T. ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium d-alanine:d-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of d-alanine. Biochemistry 27, 3709–3714 (1988).

    CAS  PubMed  Google Scholar 

  41. 41.

    Garnham, C. P., Yu, I., Li, Y. & Roll-Mecak, A. Crystal structure of tubulin tyrosine ligase-like 3 reveals essential architectural elements unique to tubulin monoglycylases. Proc. Natl Acad. Sci. USA 114, 6545–6550 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Hamano, Y., Arai, T., Ashiuchi, M. & Kino, K. NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s. Nat. Prod. Rep. 30, 1087–1097 (2013).

    CAS  PubMed  Google Scholar 

  43. 43.

    Bonnet, C. et al. Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation. J. Biol. Chem. 276, 12839–12848 (2001).

    CAS  PubMed  Google Scholar 

  44. 44.

    Boucher, D., Larcher, J. C., Gros, F. & Denoulet, P. Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin. Biochemistry 33, 12471–12477 (1994).

    CAS  PubMed  Google Scholar 

  45. 45.

    Larcher, J. C., Boucher, D., Lazereg, S., Gros, F. & Denoulet, P. Interaction of kinesin motor domains with α- and β-tubulin subunits at a Tau-independent binding site. Regulation by polyglutamylation. J. Biol. Chem. 271, 22117–22124 (1996).

    CAS  PubMed  Google Scholar 

  46. 46.

    Natarajan, K., Gadadhar, S., Souphron, J., Magiera, M. M. & Janke, C. Molecular interactions between tubulin tails and glutamylases reveal determinants of glutamylation patterns. EMBO Rep. 18, 1013–1026 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Ogasawara, Y. & Dairi, T. Biosynthesis of oligopeptides using ATP-Grasp enzymes. Chemistry 23, 10714–10724 (2017).

    CAS  PubMed  Google Scholar 

  48. 48.

    Dar, A. C. & Shokat, K. M. The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling. Annu. Rev. Biochem. 80, 769–795 (2011).

    CAS  PubMed  Google Scholar 

  49. 49.

    Schumacher, D. et al. Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew. Chem. Int. Ed. Engl. 54, 13787–13791 (2015).

    CAS  PubMed  Google Scholar 

  50. 50.

    Banerjee, A. et al. Site-specific orthogonal labeling of the carboxy terminus of α-tubulin. ACS Chem. Biol. 5, 777–785 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Google Scholar 

  52. 52.

    Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

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

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr 65, 1074–1080 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Vemu, A., Garnham, C. P., Lee, D. Y. & Roll-Mecak, A. Generation of differentially modified microtubules using in vitro enzymatic approaches. Methods Enzymol. 540, 149–166 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Widlund, P. O. et al. One-step purification of assembly-competent tubulin from diverse eukaryotic sources. Mol. Biol. Cell. 23, 4393–4401 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Banerjee, A., Bovenzi, F. A. & Bane, S. L. High-resolution separation of tubulin monomers on polyacrylamide minigels. Anal. Biochem. 402, 194–196 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D.-Y. Lee from the Biophysics Core (National Heart, Lung and Blood Institute) for access to and advice on mass spectrometry. N.T. is supported by the intramural program of the National Heart, Lung and Blood Institute (NHLBI). A.R.-M. is supported by the intramural programs of the National Institute of Neurological Disorders and Stroke (NINDS) and NHLBI. M.E.T. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

Author information

Affiliations

Authors

Contributions

K.K.M. obtained crystals, collected X-ray data, solved and refined all structures and performed functional assays and mass spectrometry analyses. E.K.K. purified proteins, obtained the wild-type ATP and elongation inhibitor crystals and collected and processed X-ray data. M.S. collected and interpreted NMR data. Y. Li collected and interpreted MS/MS data. Y. Liu synthesized inhibitors. H.L.B. synthesized branched peptides. M.E.T. supervised Y. Liu. N.T. interpreted NMR data. A.R-M. initiated, coordinated and supervised the project. K.K.M interpreted functional data. K.K.M and A.R.-M. analyzed structures and wrote the manuscript with contributions from M.S. and N.T. All authors read and approved the manuscript.

Corresponding author

Correspondence to Antonina Roll-Mecak.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Peer reviewer reports are available. Katarzyna Marcinkiewicz and Inês Chen were the primary editors 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.

Extended data

Extended Data Fig. 1 TTLL6 is an α-tubulin elongase.

a, b, Extracted-ion chromatogram of TTLL6 modified α1B443E1 peptide to which one (a) and three heavy glutamates (b) were added to the existing mono-glutamate at position E443. (ce) MS/MS sequencing of α1B445E1 peptide glutamylated by TTLL6 showing the addition of one (c), two (d) and three (e) heavy glutamates to the existing mono-glutamate at position E445. Individual b- and y- ion series and the amino acid sequence corresponding to each spectrum are indicated. Asterisks indicate ions with a neutral loss of a water molecule. (fh) Extracted-ion chromatogram of TTLL6 modified α1B445E1 peptide to which one (f), two (g) and three heavy glutamates (h) were added to the existing mono-glutamate at position E445.

Extended Data Fig. 2 TTLL6 is an α-tubulin elongase.

a, Deconvoluted α- and β-tubulin mass spectra of unmodified human microtubules glutamylated by TTLL6 (STAR Methods). The number of added glutamates is indicated and colored according to tubulin isoform. (bf) MS/MS sequencing of the α-tubulin C-terminal tails of microtubules glutamylated by TTLL6. Mono-glutamylated species in (b), di-glutamylated species in (c), tri-glutamylated species in (d), tetra-glutamylated species in (e), and penta-glutamylated species in (f). Individual b-, y-ion series and the amino acid sequence corresponding to each spectrum are indicated. m/z values of the peaks are shown in blue. Asterisks indicate ions with a neutral loss of a water molecule. (gk) Extracted-ion chromatogram of mono-glutamylated (g), di-glutamylated (h), tri-glutamylated (i), tetra-glutamylated (j) and penta-glutamylated (k) C-terminal α-tubulin tail proteolytically excised from microtubules showing modification at E443.

Extended Data Fig. 3 TTLL6 preferentially glutamylates detyrosinated α-tubulin in recombinant human microtubules.

TTLL6 glutamylation activity with recombinant α1A-Y/βIII (orange) and α1A/βIII (red) microtubules. Error bars indicate s.e.m (n=4 independent experiments) Source data (unprocessed western blots) are available online.

Source data

Extended Data Fig. 4 NMR spectroscopy confirms that the synthetic peptide used as a TTLL6 substrate has a glutamate branch at E445.

a, A zoom region of the overlaid 2D HH-TOCSY and NOESY experiments. The 2D TOCSY correlates proton spin systems. The region selected shows the HN (x-axis) and glutamate Hβ /Hγ (y-axis) correlation. Each HN strip is assigned to a residue. The 2D NOESY correlates resonances that are close in space (< ~6 Å). The highlighted peak is a NOE between the Hγ atoms of residue E445 of the tubulin main chain and the HN atom of the synthetically added 14N-Glu residue. (b) NOE highlighted in part (a) shown on the structure of the peptide.

Extended Data Fig. 5 Proposed catalytic mechanism for TTLL6 and inhibition curves for the α-elongation, γ-elongation, and initiation analogs.

(ac) Proposed catalytic mechanism for (a) α-elongating, (b) γ-elongating and (c) initiating glutamylation reactions catalyzed by TTLL6. (df) Time dependent inhibition of TTLL6 by an α-elongation analog (d), γ-elongation analog (e) and initiation analog (f). Error bars indicate s.e.m (n=4 independent experiments).

Extended Data Fig. 6 Structure of related TTLL enzymes and TTLL6 with tetrahedral intermediate analogs.

a, Cartoon representation of previously determined crystal structures of related TTLL family of enzymes. Left to right: tubulin tyrosine ligase TTL (pdb code:4IHJ), closely related glutamylase TTLL7 (pdb code:4YLR), and the glycylase TTLL3 (pdb code:5VLQ). Nucleotides are shown as stick model. Dotted lines represent regions of the polypeptide chain that are disordered in the crystal structure (b) Mechanism for inhibitor phosphorylation (c) Active site showing the |Fo|-|Fc| density (prior to modeling the γ-elongation analog) contoured at 4σ (blue). d, Active site showing the |Fo|-|Fc| density (prior to modeling the initiation analog) contoured at 3σ (blue). e, Electrostatic surface of the TTLL6 active site showing the high electropositive character and the positively charged groove adjacent to the acceptor glutamate binding site. α-elongation analog shown as a stick model. The donor glutamate, transferred phosphate and acceptor glutamate of the α-elongation analog are colored pink, orange and cyan, respectively. Conserved residues are labeled on the molecular surface.

Extended Data Fig. 7 Conservation of active site residues throughout the TTLL family.

a, Table showing conservation of critical active site residues among TTLL family members. Glutamylase initiases shown in grey, Glutamylase elongases in magenta; Glycylation initiases in green, Glycylation elongases, in blue. b, TTLL6 active site molecular surface color-coded according to conservation (as in (a)) illustrating the strong conservation in the donor glutamate binding site and variability of the acceptor glutamate binding site. c, Schematic of TTLL6 key active site interactions with the α-elongation analog.

Extended Data Fig. 8 TTLL4 is a β-tubulin specific glutamylation initiase.

a, Extracted-ion chromatogram of enzymatically added monoglutamylated, βI442E1 peptides with modifications at E438 and E439. b, Deconvoluted α- and β-tubulin mass spectra of Taxol stabilized human microtubules glutamylated by TTLL4 at a 1:10 enzyme:tubulin molar ratio after 0, 1, 2 and 4hr. The number of added glutamates is indicated and colored according to the tubulin isoform. (cf) MS-MS sequencing of the β-tubulin C-terminal tails of microtubules glutamylated by TTLL4. Mono-glutamylated species are shown in (c) and (d), di-glutamylated species in (e), tri-glutamylated species in (f). Underline signifies that the spectra are ambiguous and the third glutamate can be added to either E442 or E443. Individual b, y-ion series and the amino acid sequence corresponding to each spectrum are indicated. m/z values of the peaks are shown in blue. Asterisks indicate ions with a neutral loss of a water molecule.

Extended Data Fig. 9 TTLL6 engineered mutant functions primarily as an initiase.

MS-MS sequencing of the di-glutamylated α-tubulin C-terminal tails of microtubules glutamylated by the TTLL6 C179A/Q180R/H362I mutant showing monoglutamylation at E443 and E447 (a) and E441 and E443 (b). Individual b-, y-ion series and the amino acid sequence corresponding to each spectrum are indicated. m/z values of the peaks are shown in blue. Asterisks indicate ions with a neutral loss of a water molecule.

Extended Data Fig. 10 Inhibition curves and crystal structures for TTLL6 structure-based mutant with the α-elongation, γ-elongation, and initiation analogs.

a, Time dependent inhibition of TTLL6 C179A/Q180R/R182I/H362I/S367H mutant by the α-elongation, γ-elongation and initiation analogs. Error bars indicate s.e.m. of the fit (n=4 independent experiments). b, Active site showing the |Fo|-|Fc| density (prior to modeling the α-elongation analog) contoured at 3σ (blue). c, Active site showing the |Fo|-|Fc| density (prior to modeling the γ-elongation analog) contoured at 3σ (blue). d, Active site showing the |Fo|-|Fc| density (prior to modeling the initiation analog) contoured at 3σ (blue).

Supplementary information

Source data

Source Data Extended Data Fig. 3

Unprocessed western blots

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mahalingan, K.K., Keith Keenan, E., Strickland, M. et al. Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes. Nat Struct Mol Biol 27, 802–813 (2020). https://doi.org/10.1038/s41594-020-0462-0

Download citation

Further reading

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