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, 26 (7), 583-591

Structural Basis of Tubulin Detyrosination by Vasohibins

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Structural Basis of Tubulin Detyrosination by Vasohibins

Faxiang Li et al. Nat Struct Mol Biol.

Abstract

Microtubules are regulated by post-translational modifications of tubulin. The ligation and cleavage of the carboxy-terminal tyrosine of α-tubulin impact microtubule functions during mitosis, cardiomyocyte contraction and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1-SVBP alone, in complex with a tyrosine-derived covalent inhibitor and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α-tubulin tail by VASH1. The VASH1-SVBP-parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Fig. 1 |
Fig. 1 |. Overall structure of the human VASH1–SVBP complex.
a, Domain diagram of human VASH1 and VASH2, with the catalytic triad indicated as red lines. The domain boundaries are indicated. NTE, N-terminal extension. b, Tubulin detyrosination assays of VASH1–SVBP in human cells. Lysates of HeLa Tet-On cells transfected with the indicated plasmids and treated without or with Taxol were blotted with the indicated antibodies. deY-tubulin, detyrosinated α tubulin. Experiments were repeated three times with similar results. c, Coomassie stained gel of purified recombinant human VASH1–SVBP and VASH2–SVBP complexes. d-f, In vitro detyrosination of GMPCPP-stabilized human microtubules (MTs) (d,e), α–β tubulin heterodimer (e, bottom panels), or the C-terminal peptide of α tubulin (CTα) fused to GST (f) by the indicated recombinant VASH1–SVBP and VASH2–SVBP complexes. deY-tubulin, detyrosinated α tubulin. Experiments were repeated at least three times with similar results. g,h, Ribbon diagram of the crystal structure of the VASH152–310–SVBP complex in two different views, with the catalytic triad and S221 shown as sticks. The secondary structural elements, N- and C-termini are labeled. The color scheme of VASH1 match that in a, with the NTE, N-lobe, and C-lobe colored in light blue, blue, and cyan, respectively. SVBP is colored orange. The same color scheme is used in all subsequent figures. All structure figures were generated with PyMOL (http://www.pymol.org/). Uncropped gel and blot images of b-f are included in Supplementary Data Set 1.
Fig. 2 |
Fig. 2 |. The VASH1–SVBP interface.
a,b, Close-up views of the VASH1–SVBP interface, with interacting residues shown as sticks. VASH1 residues are colored gray and labeled with black letters while SVBP residues are colored orange and labeled with orange letters. The helices are labeled. c, Tubulin detyrosination assays of VASH1–SVBP in human cells. Lysates of HeLa Tet-On cells transfected with the indicated plasmids were blotted with the indicated antibodies. WT, wild-type; deY-tubulin, detyrosinated α tubulin. For unknown reasons, Myc-SVBP L42E and I39E/L42E mutants migrated with slower gel mobility. Uncropped blot images are included in Supplementary Data Set 1. d, Quantification of the relative detyrosination levels of α tubulin in c (mean ± SD, n = 3 independent experiments). Source data for d are available in Supplementary Data Set 2.
Fig. 3 |
Fig. 3 |. Recognition of the C-terminal tyrosine by VASH1.
a, Chemical structure of epoY. b, The 2Fo-Fc composite omit map of epoY covalently attached to VASH1 C169 (contoured at 1.0 σ, carve=1.8Å). c, Ribbon diagram of the crystal structure of the VASH1–SVBP–epoY complex, with epoY and C169 shown as sticks. d, Superimposition of the apo-VASH1–SVBP (blue and cyan ribbon) and epoY-bound VASH1–SVBP (gray ribbon). e, Close-up view of the interactions between VASH1 and epoY. f, Tubulin detyrosination assays of VASH1–SVBP in human cells. Lysates of HeLa Tet-On cells transfected with the indicated plasmids were blotted with the indicated antibodies. WT, wild-type; deY-tubulin, detyrosinated α tubulin. Uncropped blot images are included in Supplementary Data Set 1. Experiments were repeated three times with similar results. g, HeLa Tet-On cells were co-transfected with Myc-SVBP WT and the indicated VASH1 WT or mutant plasmids and stained with anti-α tubulin (red) and detyrosinated tubulin (deY-tubulin) (green) antibodies and DAPI (blue). Scale bars, 10 μm. Experiments were repeated three times with similar results.
Fig. 4 |
Fig. 4 |. Requirement for basic residues lining the substrate-binding cleft in tubulin detyrosination.
a, Ribbon diagram of the VASH1–SVBP–epoY complex with epoY and several basic residues that line the substrate-binding cleft shown as yellow and gray sticks, respectively. b, Solvent accessible surface of the VASH1–SVBP–epoY complex colored by electrostatic potential (blue, positive; red, negative). epoY is shown as sticks. c, Quantification of the relative detyrosination activities of the indicated VASH1 mutants in human cells as determined by immunoblotting (mean ± SD, n = 3 independent experiments). Representative immunoblots are shown in Supplementary Fig. 4. Source data for c are available in Supplementary Data Set 2. d, HeLa Tet-On cells were co-transfected with Myc-SVBP WT and the indicated VASH1 WT or mutant plasmids and stained with anti-α tubulin (red) and detyrosinated tubulin (deY-tubulin) (green) antibodies and DAPI (blue). Scale bars, 10 μm.
Fig. 5 |
Fig. 5 |. Mechanism of VASH1 inhibition by parthenolide.
a, Chemical structures of parthenolide before and after its covalent attachment to VASH1 C169. b, Inhibition of VASH1–SVBP-dependent tubulin detyrosination by parthenolide. HeLa Tet-On cells were co-transfected with Myc-VASH1 and Myc-SVBP plasmids and treated with increasing doses of parthenolide for 8 hours. Cell lysates were blotted with the indicated antibodies. deY-tubulin, detyrosinated α tubulin. Uncropped blot images are included in Supplementary Data Set 1. Experiments were repeated three times with similar results. c, Ribbon diagram of the crystal structure of the VASH1–SVBP–parthenolide complex, with parthenolide and C169 shown as sticks. d, The 2Fo-Fc composite omit map of parthenolide covalently linked to C169 (contoured at 1.0 σ, carve=1.8Å). e, Close-up view of the interactions between VASH1 and parthenolide. f, Model of substrate recognition and tubulin detyrosination by VASH1–SVBP and VASH2–SVBP. The “+”s indicate positive charges. The dashed line indicates unidentified interactions between VASH–SVBP and the microtubule lattice.

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