Confirmation of Allosteric Switches in Human Sirtuin 1 Using a Sirtuin Activating Compound

Abstract

Sirtuin 1 (SIRT1) is a protein deacetylase that requires cosubstrate NAD+ for its enzymatic function. A favorable target for drug development, SIRT1 is involved in stress response, metabolism, and age‐related diseases. Sirtuin activating compounds (STACs) are molecules that bind to a domain in SIRT1’s N‐terminus called the SBD (STAC Binding Domain), and change SIRT1 activity. In particular, resveratrol, an allosteric modulator of SIRT1, is one of the best‐studied STACs and is used in our experiment. This change in SIRT1 activity is due to allostery— a phenomenon where a modulator binds distal to the active site of an enzyme. Presently, the mechanism of how the N‐terminus interacts with the catalytic core to change enzyme activity is poorly understood. We hypothesized that SIRT1 contains an allosteric switch within the STAC binding domain that is the key to allosteric regulation of SIRT1. The allosteric switch is postulated to be a region of multiple residues that undergo a change in secondary structure upon allosteric interactions. The objective of this study is to predict and confirm a novel allosteric switch region in human SIRT1 (hSIRT1) using enzymatic assays with resveratrol and various peptide substrates. Using computational methods, SIRT1 residues 186–189 (TFVQ) and 190–193 (QHLM) were found to be highly ordered and thus predicted to be possible switch regions within the SBD. Mutating all four residues to serines would increase the disorder propensity of this region, which is expected to abolish the switch‐like behavior. To test this, we have cloned and purified these two serine mutations of SIRT1: SIRT1(186–189S) and SIRT1(190–193S). We performed enzyme‐coupled assays in triplicates to measure the steady‐state kinetics of these mutants to test for allosteric response to resveratrol and to ensure that they remained catalytically active. We obtained the Michaelis‐Menten parameters for the wildtype SIRT1 and the two mutated SIRT1 constructs, using Ac‐p53W as the peptide substrate. Wildtype SIRT1 showed an increase in catalytic efficiency after the addition of resveratrol. For both mutated constructs, the catalytic efficiency after adding resveratrol decreased, mostly due to an increase in KM, which is the opposite of what happens with wildtype SIRT1. This suggests that the mutations affected the allosteric network. Identifying an allosteric switch will provide insight on the mechanism of STACs with the wildtype enzyme, adding valuable information on allosteric regulation and the potential of developing drugs for age‐related diseases, stress responses, and metabolism.Support or Funding InformationSJSU Undergraduate Research Grant, California State University Program for Education and Research in Biotechnology Travel Grant Award, and NIH Grant 1SC2GM122000.