Elucidating the Mechanism for Allosteric Regulation of SIRT1 through the N-terminal Region - Summary This project aims to elucidate the mechanism for allosteric regulation of SIRT1 activity via the N-terminal domain of SIRT1, a conformationally dynamic region distal to the catalytic core. SIRT1 is an NAD+-dependent protein deacetylase which has been shown to play a significant role in many biological pathways, such as insulin secretion, tumor formation, lipid metabolism and neurodegeneration. For this reason, SIRT1 has been identified as a potential therapeutic target. This progress has been hampered by insufficient understanding of the molecular mechanism of the regulation of SIRT1 activity, as the C-terminal and N-terminal domains within SIRT1 play complicated roles in allosterically affecting SIRT1 activity. The N-terminal domain has been shown to potentiate SIRT1’s enzyme activity; this region also contains the STAC binding domain (SBD), a binding site for sirtuin activating compounds (STACs). However, there is limited in vitro biochemistry study regarding the role of the N-terminal domain in SIRT1 regulation. Our project is focused on understanding the allosteric interactions between the N-terminal domain and SIRT1’s catalytic core using three independent aims as detailed below that focus on the substrate-dependent regulation of SIRT1 by resveratrol, the regulation of SIRT1 by other STACs, and the intramolecular regulation of SIRT1 by motif A, a domain within its N-terminal region. Aim 1: Examine the role of complex stability and conformational dynamics in the substrate-sequence dependent regulation of SIRT1 by resveratrol, a well-studied STAC. We will compare the binding affinity of resveratrol to SIRT1 in the presence of different substrates, compare the stability of different SIRT1•substrate•resveratrol complexes, and compare the conformations of different SIRT1•substrate•resveratrol complexes. Aim 2: Explore if other STACs with similar or different structures as resveratrol can also regulate SIRT1 in a substrate-sequence dependent manner. We will characterize the substrate-sequence dependent effect of other STACs, namely piceatannol and SRT2104 on SIRT1 activity using enzyme activity assays and binding assays. Aim 3: Elucidate the mechanism of intramolecular SIRT1 regulation via motif A, an intrinsically disordered region in the N-terminus of SIRT1 and the role of phosphorylation in this regulation. We will use enzyme activity assays and binding assays, complemented by molecular dynamics simulations, to determine the effects of phosphorylation on motif A’s ability to regulate SIRT1 activity. Our studies will afford a more detailed understanding of the allosteric regulation of SIRT1 elicited by the N- terminal domain. This would clarify how the activity of SIRT1 is altered in various biological pathways and disease states, guiding a more targeted approach in modulating SIRT1 activity as a therapeutic method.
