Intramolecular Allosteric Regulation of SIRT1 Deacetylase Activity by the N-terminal Domain - Project Summary
This project aims to elucidate the mechanism for intramolecular regulation of SIRT1 activity by 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, where
the regulation of SIRT1 activity could combat diseases such as diabetes, cancer and
neurodegenerative diseases. 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 a complicated role 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 on the N-terminal domain and its role in SIRT1 mechanism. Our project is
focused on understanding the allosteric interactions between the N-terminal domain and SIRT1’s
catalytic core using three independent aims that focus on conformational changes, specific key
residues and binding events.
Various structures of SIRT1 complexed with different ligands have shown the SBD in different
orientations, suggesting a conformational flexibility of this domain. However, detailed studies linking the
N-terminal domain conformation with enzyme activity are lacking. We will study this relationship by
taking advantage of the substrate-specific SIRT1 regulator, resveratrol (Aim 1). A distinct allosteric
switch region within the N-terminal of SIRT1 has yet to be experimentally defined. To this end, we will
identify allosteric switch regions within the SIRT1 N-terminal domain (Aim 2). Additionally, an
intrinsically unstructured region in the N-terminal domain, motif A, has been shown to bind to the SBD
and potentiate SIRT1 activity as well, but the interaction parameters such as dissociation equilibrium
constants have not been defined. We will further investigate this binding interaction in vitro and study
the effects of phosphorylation at motif A on its ability to affect SIRT1 activity (Aim 3).
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. It would also provide insight into how other allosterically regulated enzymes
function in the cell.
