Identifying a switch‐like region within the N‐terminal domain of SIRT1

Abstract

Many proteins display conformational changes upon allosteric regulation. Oftentimes a few key residues, deemed a switch or switch-like region, are crucial in conveying these structural and functional allosteric changes. Identifying these switch-like regions not only helps us understand the nature of the allosteric regulations, it also pinpoints a “hot spot” for highly efficient targeting by small molecules or other ligands. In this study, we combine computational and biochemical methods to identify a switch-like region in the N-terminal domain of SIRT1, a lysine deacylase that plays important roles in regulating cellular pathways. Using computational methods based on the primary sequence of SIRT1, a region between residues 186-193 was predicted to exhibit switch-like behavior. Mutations were then introduced in this region of SIRT1. The resulting mutant SIRT1 constructs were tested for allosteric reactions to resveratrol, a known SIRT1 allosteric regulator, using an enzyme-coupled continuous activity assay. Some mutants remained active but exhibited no change in enzyme activity upon the addition of resveratrol, suggesting that this region is indeed a switch-like region for allosteric regulations of SIRT1. Furthermore, we have been able to identify and narrow down the specific switch-like residues within this region. Our findings suggest a method to predict switch-like regions in allosterically regulated enzymes based solely on the primary sequence of proteins. If further validated, this could be an efficient way to identify key residues in enzymes for therapeutic drug targeting and other applications.