© 2016 Nature America, Inc. All rights reserved. NATURE CHEMICAL BIOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology 1 ARTICLE PUBLISHED ONLINE: 25 JULY 2016 | DOI: 10.1038/NCHEMBIO.2140 A cetylation on lysine residues of histone and other proteins has been recognized as a major post-translational modifica- tion that affects multiple aspects of protein function. Protein acetylation levels are regulated by the balance of enzymes with opposing activities: histone acetyltransferases (HATs) and HDACs 1 . HDAC6 is the major deacetylase for tubulin, and it also deacetylates proteins such as HSP90 and cortactin, among others 2–4 . It is mostly cytoplasmic with unique characteristics that set it apart from other deacetylases: tandem catalytic domains with the capacity to deacety- late tubulin and the presence of a zinc finger domain with homology to ubiquitin-specific proteases (ZnF-UBP), which binds unanchored ubiquitin. HDAC6 is a central modulator of stress responses and autophagic clearance, essential for the formation of aggresomes or stress granules 5–7 . It also has an important role for regulatory T cells 8 , influenza virus infection 9 and in pathological conditions such as cancer, inflammation and neurodegeneration 10,11 . Microtubules (MTs) are assembled from α- and β-tubulin het- erodimers to form dynamic cytoplasmic filaments, involved in multiple cellular functions comprising cell cycle, cell shape, cellu- lar motility and intracellular transport of cargos such as vesicles or viruses 12 . MTs are heavily decorated by post-translational modifi- cations including acetylation, glutamylation, tyrosination or phos- phorylation, which have been proposed to regulate their properties, stability and functions 13,14 . α-TAT is the only acetyltransferase tar- geting Lys40, a residue located in a flexible loop of α-tubulin in the luminal side of MTs 15,16 . α-TAT prefers MTs over α/β-tubulin heterodimers for the efficient acetylation of α-tubulin Lys40 (refs. 17,18), and stochastic acetylation of MTs by α-TAT had been recently demonstrated 19 . Deacetylation of tubulin is promoted by HDAC6 (refs. 2,20) and by the NAD-dependent class III deacety- lase SIRT2 (ref. 21). HDAC6 and SIRT2 interact and may function together 21,22 . However, alteration of HDAC6 levels is sufficient to increase tubulin acetylation, and fibroblasts lacking HDAC6 have fully acetylated tubulin 23 . HDAC6 also interacts with plus-end tip-binding proteins such as EB1 or Arp1 (ref. 24), indicating that it might deacetylate the end of microtubules. It is not firmly established which is the preferred substrate of HDAC6, α/β-tubulin dimers or polymeric MTs 2,25 . It has recently been reported that inter- action between HDAC6 and tubulin is direct 26 but also that septins facilitate interaction between HDAC6 and acetylated α-tubulin 27 . HDACs comprise 11 family members and are considered prom- ising targets in a number of pathologies, with cancer being the most advanced indication 28 . In most cases however, the critical HDAC(s) have not been conclusively identified, and the four inhibitors (vor- inostat, romidepsin, belinostat and panobinostat) approved for cancer treatment until now all target multiple HDACs 29 . HDAC6 is currently evaluated as a potential therapeutic target in particu- lar in multiple myeloma. HDAC6-selective inhibitors have been developed and clinical trials are underway with a recent HDAC6- selective inhibitor, ricolinostat (ACY-1215) 30 . We solved the crystal structure of both catalytic domains of HDAC6, which together with the inter-domain linker form an ellip- soid-shaped complex with pseudo-twofold symmetry. We structur- ally and functionally defined features that are critical for HDAC6 to deacetylate its physiologic substrate tubulin, and we showed that HDAC6 prefers tubulin dimers as substrate but that it can stochasti- cally deacetylate MTs. We also determined the crystal structures of individual catalytic domains bound to either enantiomer of TSA or to the HDAC6-specific inhibitor nexturastat A (NextA), and found that (S)-TSA had moderate selectivity for HDAC6. RESULTS Organization of the HDAC6 tandem catalytic domains To understand how HDAC6 deacetylates tubulin and other substrates we characterized this multidomain protein by X-ray crystallogra- phy. As we did not obtain crystals with the mouse protein, we used the zebrafish ortholog, which efficiently deacetylates tubulin from various sources (Supplementary Results, Supplementary Fig. 1). To facilitate the comparison between zebrafish and other species, we aligned sequences of HDAC6 proteins (Supplementary Fig. 2). We first used proteins containing the tandem catalytic domains to characterize their activity in an HDAC assay using Fluor de Lys as 1 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland. 2 Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA. 3 Faculty of Sciences, University of Basel, Basel, Switzerland. 4 These authors contributed equally to this work. *e-mail: patrick.matthias@fmi.ch Structural insights into HDAC6 tubulin deacetylation and its selective inhibition Yasuyuki Miyake 1,4 , Jeremy J Keusch 1,4 , Longlong Wang 1 , Makoto Saito 1 , Daniel Hess 1 , Xiaoning Wang 2 , Bruce J Melancon 2 , Paul Helquist 2 , Heinz Gut 1 & Patrick Matthias 1,3 * 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 tan- dem 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 a-helix and a flexible tryptophan residue in the loop joining a-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measure- ments and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated a-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.