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 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.

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. Deacetylation is important for many cellular processes and aberrant KDAC activity has been linked to numerous diseases. While proper deacetylation is known to be critical for proper cellular function, details regarding substrates of particular KDACs and how substrate specificity is determined are lacking. We have observed that the activity of KDAC8 with charged substrates in vitro is sensitive to the ionic strength of the reaction buffer. This observation led us to hypothesize that charged residues near the acetylated lysine may be important for the interaction of KDAC8 with its substrates. To test this hypothesis, we have employed an in vitro assay which relies on the reaction of fluorescamine with lysine residues to produce a quantitative fluorescent signal upon deacetylation of a peptide substrate. Using this method, KDAC8 activity was measured with several substrates under conditions of varied ionic strength. Interestingly, for several positively charged peptide substrates, KDAC8 activity increases as ionic strength is decreased, but the opposite is often observed when negatively charged peptide substrates are present. Preliminary results with derivatives of a known peptide substrate suggest that a positively charged side chain adjacent to the acetylated lysine may make a specific contact with KDAC8, as the ionic strength effect varies with the position of the charge in the substrate sequence. Screening a larger set of peptides with charged residues at specific positions relative to the acetylated lysine for activity with KDAC8 provides insight into the contributions of charge to substrate specificity. Comparison of these data with molecular docking data of the same peptides with the active site of KDAC8 can predict specific interactions between charged residues in the KDAC8‐substrate pair. Together, these results will lead to a better understanding of KDAC8 substrate specificity. Extension of this approach to additional KDACs and substrate behavior in vivo will provide insight into substrate specificity of the KDAC family and the biological targets of each KDAC.Support or Funding InformationFunding was provided by NIH 5G12MD007595, R15GM129682, TL4GM118968, 5RL5GM118966, and the Louisiana Cancer Research Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.