Identifying an Allosteric Switch Region in Human SIRT1

Abstract

SIRT1 is an NAD+‐dependent lysine deacetylase that is implicated in age‐related diseases and is a potential therapeutic target for drug development. The N‐terminal domain is highly disordered and is usually removed prior to crystallization of the enzyme. However, it has been known to physically interact with the catalytic core to increase enzyme activity. The mechanism of this remains unknown. STAC binding domain (SBD), within the N‐terminal region, is the binding site for sirtuin‐activating compounds (STACs) such as resveratrol, which is known to enhance SIRT1 activity towards peptide substrates including p53W. An allosteric switch is hypothesized as a single or multiple residues responsible for a change in secondary structure essential for allosteric activity and mostly found in disordered regions. Detailed information on switch regions remains unexplored due to the challenges of crystallizing the disordered regions. Our project aims to identify and confirm an allosteric switch region within the N‐terminal domain of SIRT1. The first aim is to computationally identify a switch region in SIRT1 using sequence‐and homology‐based descriptors. A possible switch region is hypothesized as having (1) high secondary structure variability, (2) low sequence entropy, and (3) low sequence disorder propensity, inferred by a combination of predictors. Applying these predictors to the full sequence of SIRT1 showed that such a switch may occur within residues 186–193 (TFVQQHLM) of the SBD. The second aim is to experimentally confirm these predictions. We have mutated the predicted region to serine residues: 186–189S, 190–193S, 188S, and 192S. The enzyme kinetics of the mutants remain relatively similar to the wild‐type SIRT1. We then tested these mutants for susceptibility to allosteric modulations by assaying their enzyme activity towards p53W with and without resveratrol. No increase in activity with the addition of resveratrol would confirm that these residues are involved in an allosteric switch. These results will inform the accuracy of the computational descriptors and provide a better understanding of the regulation of this enzyme in the cellular system.Support or Funding InformationCalifornia State University Program for Education and Research in Biotechnology; Louis Stokes Alliance for Minority ParticipationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.