Abstract
Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.
Highlights
Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins
We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40 –300-fold higher than those for corresponding peptide substrates
The addition of DNA to form mononucleosomes decreases reactivity with HDAC8. These results demonstrate that HDAC8 specificity for H3 peptide tetramer substrates is not determined solely based on the six amino acids proximal to the acetyl-lysine; substrate specificity of HDAC8 is modulated by both long-range and short-range contacts for H3 substrates
Summary
We focused on the activity of HDAC8 with three acetylated lysine sites within histone H3, a histone known to be amenable to non-natural acetyl-lysine incorporation [54]. Two H3 acetylation sites (H3K9ac and H3K14ac) are located within proximity to each other on the N-terminal tail and share an unfolded secondary structure Because these sites differ only in amino acid sequence, the role of primary sequence in HDAC8 substrate specificity can be probed. The rates of HDAC8-catalyzed deacetylation of 7-mer peptides representing the three amino acids upstream and downstream of the H3K9ac, H3K14ac, and H3K56ac acetylation sites were measured under multiple turnover (MTO) conditions, using an assay coupling acetyl-lysine deacetylation to the formation of NADH (Table 1) [55]. To probe the importance of amino acids at further distances from the acetyl-lysine in determining substrate selectivity, longer peptides (13 and 17 amino acids) were assayed (Table 1). HDAC8 activity was measured, and catalytic efficiencies were determined as described under “Experimental procedures” and the legends of Figs. 2– 4
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