Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene.

Sirtuin isoform 2 (SIRT2) is an enzyme that catalyzes the removal of acyl groups from lysine residues. SIRT2's catalytic domain has a hydrophobic tunnel where its substrate acyl groups bind. Here, we report that the fluorescent probe 1-aminoanthracene (AMA) binds within SIRT2's hydrophobic tunnel in a substrate-dependent manner. AMA's interaction with SIRT2 was characterized by its enhanced fluorescence upon protein binding (>10-fold). AMA interacted weakly with SIRT2 alone in solution (Kd = 37 μM). However, when SIRT2 was equilibrated with a decanoylated peptide substrate, AMA's affinity for SIRT2 was enhanced ∼10-fold (Kd = 4 μM). The peptide's decanoyl chain and AMA co-occupied SIRT2's hydrophobic tunnel when bound to the protein. In contrast, binding of AMA to SIRT2 was competitive with a myristoylated substrate whose longer acyl chain occluded the entire tunnel. AMA competitively inhibited SIRT2 demyristoylase activity with an IC50 of 21 μM, which was significantly more potent than its inhibition of other deacylase activities. Finally, binding and structural analysis suggests that the AMA binding site in SIRT2's hydrophobic tunnel was structurally stabilized when SIRT2 interacted with a decanoylated or 4-oxononanoylated substrate, but AMA's binding site was less stable when SIRT2 was bound to an acetylated substrate. Our use of AMA to explore changes in SIRT2's hydrophobic tunnel that are induced by interactions with specific acylated substrates has implications for developing ligands that modulate SIRT2's substrate specificity.