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. 2017 Sep 14;7:11547. doi: 10.1038/s41598-017-11739-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Structural features outside the αK40 loop are required for efficient substrate deacetylation by HDAC6. (a,b) Michaelis-Menten kinetics for peptides derived from the Lys40 of α-tubulin (αK40) loop (3-mer to 19-mer; T3 through T19) were determined in vitro using an HPLC-based assay (a). Corresponding kinetic parameters derived from the non-linear regression fit of experimental data, together with peptide sequences, are shown in (b). High micromolar K _M values indicate low affinity of HDAC6 for tested peptides, with limited contribution of residues that do not directly neighbor the central acetyllysine for the overall HDAC6 affinity/specificity. (c) Comparison of HDAC6 deacetylation rates using various substrates (natively-folded αβ-tubulin dimers, denatured tubulin dimers, peptides T3 through T19) at identical 3 μM concentrations. Deacetylation rates were determined using an HPLC assay for peptides and Western blotting quantification for tubulin dimers. Natively folded tubulin dimers are deacetylated 50- to 800-fold more efficiently than are either denatured tubulin dimers or “isolated” αK40 loop-derived peptides, suggesting that interactions/structural features outside the αK40 loop are required for efficient HDAC6/tubulin interactions. Data are presented as mean values ± s.d. (n = 3). (d) Steady-state kinetics of tubulin dimer deacetylation by HDAC6. Michaelis-Menten constants (K_M and k _cat) were calculated from non-linear regression fit using the GraphPad program. Data represent mean values ± s.d. (n = 3).