Allosteric Modulation of SIRT1 Activity Affects Substrate Recognition and Protein Conformation

Abstract

SIRT1 is a lysine deacetylase that plays an important role in regulating various cellular pathways such as neurodegeneration and insulin secretion. As such, it is of great interest to modulate SIRT1 activity as a therapeutic approach for diseases such as Alzheimer’s disease and diabetes. Several small‐molecule allosteric regulators of SIRT1, known as STACs (Sirtuin Activating Compounds) are studied, such as resveratrol. It is known that these regulators bind to the STAC Binding Domain (SBD) of SIRT1, distal from the enzyme active site, and regulates SIRT1 activity through allosteric effects. However, the molecular mechanism of these allosteric effects is not well understood, impeding more specific designs of SIRT1 modulator compounds. In this study we take a systematic approach to study the changes in Michaelis‐Menten kinetics and overall conformation of SIRT1 when its activity is allosterically decreased and increased. A previous end‐point screen has found that the effect of resveratrol on SIRT1 is substrate‐sequence dependent, where resveratrol can increase, decrease, or have no effect on the activity of SIRT1 against different subsets of peptide substrates. We have chosen several peptides from each of the above‐mentioned categories and determined the Michaelis‐Menten kinetics of SIRT1 against these substrates with and without resveratrol. Upon comparison, we find that the allosteric effect of resveratrol mainly changes the substrate recognition (KM) of SIRT1 for these peptides. In addition, we used Small Angle X‐Ray Scattering (SAXS) to monitor the overall conformation of SIRT1 in complex with the different peptide substrates with and without resveratrol and found that the protein conformation is altered upon addition of resveratrol. Combining these findings, we have a better understanding of the mechanism for allosteric regulation of SIRT1, which could shed light on other allosteric regulations in the cell and aide in guided design of small molecule SIRT1 modulators.Support or Funding InformationNational Institutes of Health NIGMS SC2GM132000‐01