Abstract 1645 Characterization of the dark kinase DCLK3 reveals isoform-specific modification and novel substrates

Metal-dependent lysine deacetylases (KDACs, also known as histone deacetylases or HDACs) are a family of enzymes responsible for reversing the post-translation modification of lysine acetylation.Because the eleven members of the KDAC family have a conserved catalytic domain structure, the determinants of substrate selectivity for each KDAC are not obvious.Although some acetylated proteins have been associated with particular KDACs as putative substrates, there are few such proteins for which the KDAC responsible for deacetylation has been definitively established.Similarly, many KDAC inhibitors have been identified, but there has been limited success in designing inhibitors that selectively target the KDAC family or a single KDAC.Therefore, there is a need to better understand the interactions between KDACs and other molecules.We have previously identified a few features in some of the KDACs that contribute to the unique substrate selectivity of each enzyme.Here, we expand on that work to present an interaction map of the entire KDAC8 binding surface, identifying the key interactions that drive substrate selectivity.Catalytic activity of KDAC8 was assessed using a library of acetylated peptides, primarily derived from the sequences of proteins with observed acetylation.Molecular dynamics simulations were used to identify interactions between KDAC8 surface residues and peptide residues for each member of the library.Correlations between the activity profile and interaction profiles were used to identify potentially important interactions determining substrate preferences.Those interactions were validated both experimentally and computationally using mutated enzyme variants.Overall, we have created a specificity profile of KDAC8 and identified the molecular interactions driving that specificity.Furthermore, we have made progress toward identifying which of the interactions are specific to KDAC8 and which are also features of other members of the KDAC family, as well as demonstrating that commonly utilized inhibitors fail to target these interactions.The specificity profile can be applied to identify likely biological targets, while the profile of characteristic molecular interactions can be applied when designing and evaluating molecules targeting one or more members of the KDAC family.