In the cell, tubulin is post-translationally modified to create functionally distinct microtubules (MTs) endowed with specialized functions. Acetylation of Lys-40 of α-tubulin (αK40) is one of major post-translational modifications that provides long-lived MTs with mechanical resilience. The αK40 level is controlled by histone deacetylase 6 (HDAC6), a multidomain cytosolic protein that acts as a major tubulin deacetylase. While the tubulin deacetylase activity of HDAC6 has been unequivocally assigned to the second of the duplicated catalytic domains, there is virtually no information on contribution of other domains on HDAC6/tubulin interactions and deacetylation efficacy.
Using deletion mutagenesis, we identified the N-terminal part of human HDAC6 as a microtubule-binding (MTB) domain and functionally characterized it up to the single molecule level. We show that the MTB domain is solely responsible for 100 nM HDAC6 affinity for stabilized MTs and the MT-binding motif spans a positively charged patches comprising amino acids 32 – 37 and 51 – 56. HDAC6/MT interactions are fully independent of the presence catalytic domains and are mediated by ionic interactions with C-terminal unstructured tubulin tails. At the same time, the interplay between the MTB and deacetylase domains is critical for recognition and efficient deacetylation of tubulin, but not small peptidic substrates. Overall, our data reveal that recognition of natural substrates by HDAC6 is more complex than previously appreciated and domains outside the tandem catalytic core are essential for proficient substrate deacetylation in vivo.