Evolved, Selective Erasers of Distinct Lysine Acylations

Abstract

Lysine acetylation, including related lysine modifications such as butyrylation and crotonylation, is a widespread post-translational modification with important roles in many important physiological processes. However, uncovering the regulatory mechanisms that govern the reverse process, deacylation, has been challenging to address, in great part because the small set of lysine deacetylases (KDACs) that remove the modifications are promiscuous in their substrate and acylation-type preference. This lack of selectivity hinders a broader understanding of how deacylation is regulated at the cellular level and how it is correlated with lysine deacylation-related diseases. To facilitate the dissection of KDACs with respect to substrate specificity and modification type, it would be beneficial to re-engineer KDACs to be selective towards a given substrate and/or modification. To dissect the differential contributions of various acylations to cell physiology, we developed a novel directed evolution approach to create selective KDAC variants that are up to 400-fold selective towards butyryl- over crotonyl-lysine substrates. Structural analyses of this non-promiscuous KDAC revealed unprecedented insights regarding the conformational changes mediating the gain in specificity. As a second case study to illustrate the power of this approach, we re-engineer the human SirT1 to increase its selectivity towards acetylated versus crotonylated substrates. These new enzymes, as well as the generic approach that we report here, will greatly facilitate the dissection of the differential roles of lysine acylation in cell physiology.\n\nSignificance StatementAcetylation of lysine residues features numerous roles in diverse physiological processes and correlates with the manifestation of metabolic diseases, cancer and ageing. The already huge diversity of the acetylome is multiplied by variations in the types of acylation. This complexity is in stark contrast to the small set of lysine deacetylases (KDACs) present in human cells, anticipating a pronounced substrate promiscuity.\n\nWe device a strategy to tackle this disarray by creating KDAC variants with increased selectivity towards particular types of lysine acylations using a novel selection system. The variants facilitate the dissection of the differential contributions of particular acylations to gene expression, development and disease. Our structural analyses shed light on the mechanism of substrate discrimination by Sirtuin-type KDACs.