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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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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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Contributions

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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