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

Nature Structural & Molecular Biology volume 27, pages 802–813 (2020)Cite this article

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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.

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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.

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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

Authors and Affiliations

  1. Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA

    Kishore K. Mahalingan, E. Keith Keenan & Antonina Roll-Mecak

  2. Biochemistry & Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD, USA

    Madeleine Strickland, Nico Tjandra & Antonina Roll-Mecak

  3. Mass Spectrometry Core Facility, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA

    Yan Li

  4. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada

    Yanjie Liu & Martin E. Tanner

  5. University of Texas Southwestern Medical Center, Protein Chemistry Technology Center, Dallas, USA

    Haydn L. Ball

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  1. Kishore K. Mahalingan
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  4. Yan Li
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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.

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Correspondence to Antonina Roll-Mecak.

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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. (c–e) 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. (f–h) 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. (b–f) 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. (g–k) 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.

(a–c) Proposed catalytic mechanism for (a) α-elongating, (b) γ-elongating and (c) initiating glutamylation reactions catalyzed by TTLL6. (d–f) 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. (c–f) 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).

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Source data

Source Data Extended Data Fig. 3

Unprocessed western blots

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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

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