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Structural insights into HDAC6 tubulin deacetylation and its selective inhibition

Abstract

We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.

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Figure 1: Overall structure of HDAC6 catalytic domains.
Figure 2: Conservation analysis of CD1 vs.CD2.
Figure 3: Structural and molecular determinants for tubulin deacetylation.
Figure 4: HDAC6 prefered tubulin dimers, but deacetylated MTs stochastically.
Figure 5: HDAC6-specific inhibitor binding.

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References

  1. Yang, X.J. & Seto, E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol. 9, 206–218 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Kovacs, J.J. et al. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell 18, 601–607 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Zhang, X. et al. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell 27, 197–213 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Boyault, C. et al. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev. 21, 2172–2181 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kawaguchi, Y. et al. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727–738 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Kwon, S., Zhang, Y. & Matthias, P. The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 21, 3381–3394 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. de Zoeten, E.F. et al. Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp3(+) T-regulatory cells. Mol. Cell. Biol. 31, 2066–2078 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Banerjee, I. et al. Influenza A virus uses the aggresome processing machinery for host cell entry. Science 346, 473–477 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Aldana-Masangkay, G.I. & Sakamoto, K.M. The role of HDAC6 in cancer. J. Biomed. Biotechnol. 2011, 875824 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Seidel, C., Schnekenburger, M., Dicato, M. & Diederich, M. Histone deacetylase 6 in health and disease. Epigenomics 7, 103–118 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Lawson, J.L.D. & Carazo Salas, R.E. Microtubules: greater than the sum of the parts. Biochem. Soc. Trans. 41, 1736–1744 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Janke, C. & Bulinski, J.C. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat. Rev. Mol. Cell Biol. 12, 773–786 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Yu, I., Garnham, C.P. & Roll-Mecak, A. Writing and Reading the Tubulin Code. J. Biol. Chem. 290, 17163–17172 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nogales, E., Whittaker, M., Milligan, R.A. & Downing, K.H. High-resolution model of the microtubule. Cell 96, 79–88 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Soppina, V., Herbstman, J.F., Skiniotis, G. & Verhey, K.J. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules. PLoS One 7, e48204 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kormendi, V., Szyk, A., Piszczek, G. & Roll-Mecak, A. Crystal structures of tubulin acetyltransferase reveal a conserved catalytic core and the plasticity of the essential N terminus. J. Biol. Chem. 287, 41569–41575 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shida, T., Cueva, J.G., Xu, Z., Goodman, M.B. & Nachury, M.V. The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl. Acad. Sci. USA 107, 21517–21522 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Szyk, A. et al. Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell 157, 1405–1415 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, Y. et al. HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J. 22, 1168–1179 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. North, B.J., Marshall, B.L., Borra, M.T., Denu, J.M. & Verdin, E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11, 437–444 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Nahhas, F., Dryden, S.C., Abrams, J. & Tainsky, M.A. Mutations in SIRT2 deacetylase which regulate enzymatic activity but not its interaction with HDAC6 and tubulin. Mol. Cell. Biochem. 303, 221–230 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Zhang, Y. et al. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol. Cell. Biol. 28, 1688–1701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zilberman, Y. et al. Regulation of microtubule dynamics by inhibition of the tubulin deacetylase HDAC6. J. Cell Sci. 122, 3531–3541 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Matsuyama, A. et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 21, 6820–6831 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhao, Z., Xu, H. & Gong, W. Histone deacetylase 6 (HDAC6) is an independent deacetylase for alpha-tubulin. Protein Pept. Lett. 17, 555–558 (2010).

    Article  CAS  PubMed  Google Scholar 

  27. Ageta-Ishihara, N. et al. Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation. Nat. Commun. 4, 2532 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Falkenberg, K.J. & Johnstone, R.W. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 13, 673–691 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Schölz, C. et al. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat. Biotechnol. 33, 415–423 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Santo, L. et al. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma. Blood 119, 2579–2589 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zou, H., Wu, Y., Navre, M. & Sang, B.C. Characterization of the two catalytic domains in histone deacetylase 6. Biochem. Biophys. Res. Commun. 341, 45–50 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Zhang, Y., Gilquin, B., Khochbin, S. & Matthias, P. Two catalytic domains are required for protein deacetylation. J. Biol. Chem. 281, 2401–2404 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Ouyang, H. et al. Protein aggregates are recruited to aggresome by histone deacetylase 6 via unanchored ubiquitin C termini. J. Biol. Chem. 287, 2317–2327 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Finnin, M.S. et al. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401, 188–193 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Lombardi, P.M., Cole, K.E., Dowling, D.P. & Christianson, D.W. Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes. Curr. Opin. Struct. Biol. 21, 735–743 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schuetz, A. et al. Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity. J. Biol. Chem. 283, 11355–11363 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yoshida, M., Hoshikawa, Y., Koseki, K., Mori, K. & Beppu, T. Structural specificity for biological activity of trichostatin A, a specific inhibitor of mammalian cell cycle with potent differentiation-inducing activity in Friend leukemia cells. J. Antibiot. (Tokyo) 43, 1101–1106 (1990).

    Article  CAS  Google Scholar 

  38. Yoshida, M., Kijima, M., Akita, M. & Beppu, T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265, 17174–17179 (1990).

    CAS  PubMed  Google Scholar 

  39. Cosner, C.C. et al. Evolution of Concise and Flexible Synthetic Strategies for Trichostatic Acid and the Potent Histone Deacetylase Inhibitor Trichostatin A. Eur. J. Org. Chem. 162–172 (2013).

    Article  CAS  Google Scholar 

  40. Bergman, J.A. et al. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. J. Med. Chem. 55, 9891–9899 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dowling, D.P., Gantt, S.L., Gattis, S.G., Fierke, C.A. & Christianson, D.W. Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors. Biochemistry 47, 13554–13563 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Butler, K.V. et al. Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A. J. Am. Chem. Soc. 132, 10842–10846 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Silvestri, L., Ballante, F., Mai, A., Marshall, G.R. & Ragno, R. Histone deacetylase inhibitors: structure-based modeling and isoform-selectivity prediction. J. Chem. Inf. Model. 52, 2215–2235 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Black, M.M., Baas, P.W. & Humphries, S. Dynamics of alpha-tubulin deacetylation in intact neurons. J. Neurosci. 9, 358–368 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cueva, J.G., Hsin, J., Huang, K.C. & Goodman, M.B. Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules. Curr. Biol. 22, 1066–1074 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Topalidou, I. et al. Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization. Curr. Biol. 22, 1057–1065 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D. Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Bricogne, G. et al. BUSTER version 2.11.4. (Global Phasing Ltd., Cambridge, UK, 2011).

  55. Schrödinger, LLC. The PyMOL Molecular Graphics System Version 1.7.6 (2010).

  56. Söding, J., Biegert, A. & Lupas, A.N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Martí-Renom, M.A. et al. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291–325 (2000).

    Article  PubMed  Google Scholar 

  58. Cosner, C.C. & Helquist, P. Concise, convergent syntheses of (±)-trichostatin A utilizing a Pd-catalyzed ketone enolate α-alkenylation reaction. Org. Lett. 13, 3564–3567 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Ziółkowska, N.E. & Roll-Mecak, A. In vitro microtubule severing assays. Methods Mol. Biol. 1046, 323–334 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Carbajal, A., Chesta, M.E., Bisig, C.G. & Arce, C.A. A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype. Biochem. J. 449, 643–648 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank J. Spector and A. Roll-Mecak for sharing information about total internal reflection fluorescence microscopy experiments and discussions, S. Weiler and U. Schopf and U. Rass for helpful discussions, K. Verhey for a technical suggestion, L. Gelman for help with microscopy analysis, and D. Klein and J. Seebacher for mass spectrometry analysis to identify domain boundaries. Part of this work was performed at beamlines X10SA and X06DA of the Swiss Light Source. We thank G. Matthias, C. Cao and M. Regenass for helpful technical assistance, and O. Truee, R.G. Clerc and all the members of the Matthias laboratory for fruitful discussions. This work was supported by the Novartis Research Foundation and M.S. was also partly supported by a Fellowship from the Nakajima Foundation. Work performed at the University of Notre Dame is supported by the Ara Parseghian Medical Research Foundation, the National Institutes of Health (1R01NS092653), the Warren Family Center for Drug Discovery and Development and the Department of Chemistry and Biochemisry. X.W., B.J.M. and P.H. thank J. Zajicek and J. Pontius for NMR microscopy support and discussion.

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Y.M., H.G. and P.M. designed experiments; Y.M. performed biochemical and microscopy experiments, Y.M., J.J.K., L.W. and M.S. prepared and purified proteins and performed assays; M.S. and L.W. performed cellular inhibitor assays; J.J.K. crystallized proteins; X.W., B.J.M. and P.H. synthesized and purified (R)- and (S)-TSA; H.G. and J.J.K. collected diffraction data and H.G. determined crystal structures; D.H. analyzed mass spectrometry data; Y.M., H.G. and P.M. wrote the manuscript; P.M. oversaw the work. All authors contributed to the final manuscript.

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Correspondence to Patrick Matthias.

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Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–21. (PDF 40231 kb)

Supplementary Dataset 1

ConSurf conservation scores. (XLSX 29 kb)

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Miyake, Y., Keusch, J., Wang, L. et al. Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat Chem Biol 12, 748–754 (2016). https://doi.org/10.1038/nchembio.2140

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