The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation

• Published in: The Journal of biological chemistry Vol. 295; no. 9; pp. 2614 - 2628
• Main Authors: Ustinova, Kseniya, Novakova, Zora, Saito, Makoto, Meleshin, Marat, Mikesova, Jana, Kutil, Zsofia, Baranova, Petra, Havlinova, Barbora, Schutkowski, Mike, Matthias, Patrick, Barinka, Cyril
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.02.2020; American Society for Biochemistry and Molecular Biology
• Subjects: Acetylation; Amino Acid Sequence; Catalytic Domain; cytoskeleton; Enzymology; histone deacetylase 6 (HDAC6); Histone Deacetylase 6 - metabolism; Humans; intrinsically disordered protein; microtubule-associated protein (MAP); Microtubules - metabolism; post-translational modification; Protein Binding; Protein Domains - physiology; protein motif; protein-protein interaction; structure-function; Substrate Specificity; total internal reflection fluorescence (TIRF); tubulin; Tubulin - metabolism; microtubule-associated protein (MAP); total internal reflection fluorescence (TIRF); intrinsically disordered protein; histone deacetylase 6 (HDAC6); protein motif; cytoskeleton; substrate specificity; structure-function; tubulin; protein-protein interaction; post-translational modification
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.011243

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo. Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.