Lysine acetylation and deacetylation are critical for regulation of many cellular proteins. Despite the importance of this cycle, it is unclear how lysine deacetylase (KDAC) family members discriminate between acetylated proteins to react with a discrete set of substrates. Potential short-range interactions between KDAC8 and a known biologically relevant peptide substrate were identified using molecular dynamics (MD) simulations. Activity assays with a panel of peptides derived from this substrate supported a putative ionic interaction between arginine at the −1 substrate position and KDAC8 D101. Additional assays and MD simulations confirmed this novel interaction, which promotes deacetylation of substrates. Verification that a negatively charged residue at the 101 position is necessary for the ionic interaction and observed reactivity with the substrates was performed using KDAC8 derivatives. Notably, this interaction is specific to KDAC8, as KDAC1 and KDAC6 do not form this interaction and each KDAC has a different specificity profile with the peptide substrates, even though all KDACs could potentially form ionic interactions. When reacted with a panel of putative human KDAC substrates, KDAC8 preferentially deacetylated substrates containing an arginine at the −1 position. KDAC8 D101-R(−1) is a specific enzyme−substrate interaction that begins to explain how KDACs discriminate between potential substrates and how different KDAC family members can react with different subsets of acetylated proteins in cells. This multi-pronged approach will be extended to identify other critical interactions for KDAC8 substrate binding and determine critical interactions for other KDACs.

Lysine deacetylases (KDACs) are enzymes that remove an acetyl group from a lysine, thereby regulating the post‐translational modification of lysine. KDACs are important in many different biological processes and are associated with various mechanisms of diseases in the human body. However, factors contributing to substrate specificity of the KDACs are poorly understood. Moreover, many potential substrate proteins contain adjacent or nearby lysine residues that can be independently acetylated and deacetylated. We hypothesized that KDACs will demonstrate preferences for a particular acetyllysine when reacting with multiply acetylated substrates. We additionally hypothesized that KDAC activity would exhibit a dependence on the acetylation status of nearby lysine residues. We identified several proteins reported as putative substrates of KDACs and which contain multiple nearby lysines, each of which has been reported as acetylated. Using peptides derived from these proteins, we measured activity of the putative substrates with each KDAC via a fluorescence‐based assay. The acetyllysine(s) targeted by each specific KDAC was determined by mass spectrometry. Molecular dynamics was used to model the interactions of the peptides with the KDACs. Our results demonstrate that nearby acetylation does influence the ability of individual KDACs to deacetylate a particular lysine residue, in a manner specific to each KDAC. We propose distinct contacts in the KDACs that may contribute to this selectivity. Future plans include evaluating the enzymatic activity of the KDACs with the full‐length putative substrates in cells to more firmly establish biological relevance.

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.