Specificity Determinants of Lysine Deacetylases

Abstract

Lysine acetylation is a reversible post‐translational modification that has been found on thousands of nuclear and cytoplasmic proteins. A family of enzymes, known as lysine deacetylases (KDACs), catalyzes the removal of acetyl groups. This reversible modification is a regulatory mechanism for proteins involved in numerous cellular processes; however, few KDAC‐substrate pairs have been identified, and there is a lack of information regarding how individual KDACs interact with substrates. We hypothesized that differences between KDACs near the active site would influence substrate specificity, allowing members of the family to preferentially react with a unique subset of acetylated proteins. Using a peptide library derived from a previously‐identified substrate of KDAC8, we demonstrated that KDAC1, KDAC6, and KDAC8 have distinct preferences regarding the residues surrounding the acetylated lysine. A combination of activity data using an in vitro assay and molecular dynamics simulations revealed discrete features of the KDACs that contribute to substrate specificity. In KDAC8, but not in other KDACs, an aspartic acid residue (D101) forms an ionic interaction with positively charged residues in the ‐1 position of the substrate relative to the acetyllysine, promoting deacetylation. Additionally, nearby hydrophobic residues further influence substrate preferences in a KDAC‐specific manner. Using interaction models based on these data, we were able to predict biologically relevant peptide substrates for individual KDACs. While the identified residues are undoubtedly not the only KDAC specificity determinants, understanding the role of these close contacts on enzyme preferences has greatly increased our understanding of KDAC specificity and has given us a handle to identify potential protein substrates from cells.