Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol

Abstract

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.

Keywords: histone/protein deacetylase; resveratrol; sirtuins; structure.

Figure 1.

SIRT1 fragments and their resveratrol-dependent deacetylation activities. (A) A schematic diagram showing functional domains in full-length SIRT1 (top) and a truncation variant (SIRT1-143) used for crystallization (bottom). (B) A bar diagram showing deacetylase activities of the indicated SIRT1 fragments and the stimulatory effects of 0.5 mM resveratrol. (C) The p53-AMC peptide used for cocrystallization and deacetylation assays. The single-letter residue symbol together with the residue number indicates SIRT1 residues involved in protein–peptide interactions via main chain (filled circles) or side chain (filled boxes) groups. The red dashed lines denote hydrogen bonds, the brown thick curve indicates the acetyl-lysine-binding pocket in SIRT1-CD, and the nearby indicated residues represent ones contacting the acetyl-lysine via hydrophobic and/or van der Waals interactions. Cyan and green filled shapes indicate residues in CD and NTD, respectively.

Figure 2.

Structure of the SIRT1 ternary complex. (A) Overall structure of SIRT1 with the protein domains color-coded as in Figure 1A. A pair of SIRT1 residues involved in NTD–CD interdomain interactions are shown in a stick model, with the hydrogen bonds indicated by white dashed lines. The acetylated p53-AMC peptide (carbon colored wheat) and resveratrol molecules (carbon colored magenta) are shown as a stick model. (B) A detailed view of protein–resveratrol and resveratrol–peptide interactions. (C) Superposition of SIRT1 and Sir2 structures aligned via their CDs. The ribbon structure of Sir2 is shown in light pink, and the structure of the SIRT1 complex is color-coded the same as in A, with the exception that the p53-AMC peptide is shown as a light-blue stick model. The SIR4 structure was removed for viewing clarity, but the blue-shaded region indicates its general location. (D) Superposition of the N-terminal helical domains of SIRT1 and Sir2.

Figure 3.

Comparison of resveratrol-binding modes. (A) A surface representation (with electrostatic potential distribution) of SIRT1 with the bound p53-AMC peptide and resveratrol. The NTD of SIRT1 was removed for viewing clarity. (B) The structure of SIRT5 with the bound peptide and resveratrol (Protein Data Bank [PDB] ID 4HDA). (C) The structure of SIRT3 with the bound peptide and bromo-resveratrol (PDB ID 4C7B). (D) The structure of SIRT3 with the bound piceatannol (PDB ID 4HD8). Both the peptides (carbon colored gold) and resveratrol or its analog (carbon colored green) are shown as a stick model.

Figure 4.

Deacetylase activities and peptide-binding affinities of SIRT1 variants. (A) Deacetylation rates of the indicated SIRT1 variants toward p53-AMC in the absence and presence of 0.2 mM resveratrol. (B–G) ITC measurements of binding affinities of the p53-AMC peptide to the indicated SIRT1 variants in the presence of 0.5 mM resveratrol.

Figure 5.

Resveratrol effect on native peptides. (A–D), Titration curves for determination of K_m values of SIRT1-143CS toward the indicated peptides in the absence (black curves) and presence (red curves) of 0.2 mM resveratrol. An enzyme concentration of 200 nM in B, as opposed to 400 nM in the rest, was used to slow down the reaction for better precision measurement. (E) A summary of measured Michaelis-Menten kinetic parameters for the indicated peptides in the absence and presence of 0.2 mM resveratrol. (F) Deacetylase activities of SIRT1 fragments 143-CS and 89–747 toward the indicated peptides measured at 400 nM and 50 μM enzyme and peptide concentrations, respectively, in the absence and presence of 0.2 mM resveratrol. (G) Superposition of the fluorophoreless acetylated p53 peptide (carbon colored yellow) from an archaeal sirtuin complex (PDB ID 1MA3) with the SIRT1 ternary complex. Residue positions with respect to the acetylated lysine are indicated.