Structure-based mechanism of ADP-ribosylation by sirtuins

Abstract

Sirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD(+)-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. We have studied the ADP-ribosylation activity of a bacterial sirtuin, Sir2Tm, and show that acetylated peptides containing arginine or lysine 2 residues C-terminal to the acetyl lysine, the +2 position, are preferentially ADP-ribosylated at the +2 residue. A structure of Sir2Tm bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide. The new biochemical and structural studies presented here provide mechanistic insights into the Sir2 ADP-ribosylation reaction and will aid in identifying substrates of this reaction.

FIGURE 1.

Sirtuin-mediated ribosylation of acetyl p53 peptides. A, the methionine of the acetyl p53 peptide, which is two amino acids C-terminal to the acetyl lysine, points toward the S-alkylamidate intermediate in the Sir2Tm-S-alkylamidate intermediate structure. B, acetyl p53 peptides with different amino acids two positions C-terminal to the acetyl lysine were reacted with Sir2Tm and [³²P]NAD⁺, and the reactions were resolved by SDS-PAGE gels. (+2)M, (+2) Met; (+2)E, (+2) Glu; (+2)R, (+2) Arg; (+2)K, (+2) Lys. C, ribosylation activity for the reactions from B was quantified by densitometry. Error bars indicate S.D. D, Sir2Tm was reacted with various concentrations of acetyl p53 Arg peptide (B), and the data were fit to the Michaelis-Menten model. Error bars indicate S.D. E, SIRT1 was reacted with the p53 acetyl peptides described in B and [³²P]NAD⁺. The reactions were separated by SDS-PAGE gels, and SIRT1 ribosylation activity was quantified by densitometry. Error bars indicate S.D.

FIGURE 2.

Sir2Tm ribosylation of peptides containing arginine at different positions. Sir2Tm was incubated with [³²P]NAD⁺ and different peptides that have arginine substituted at different positions in the acetyl p53 peptide sequence. A, the autoradiograph of the reactions containing Sir2Tm-, [³²P]NAD⁺-, and p53-based peptides. R indicates arginine; no ac, without acetyl; no R, without arginine. B, quantification of A by densitometry based on data from three replicates of the reaction. Error bars indicate average deviation C, sequences of the peptides used in A.

FIGURE 3.

Sir2Tm directly ribosylates the arginine two amino acids C-terminal to acetyl lysine. Manual Edman degradation was performed on the acetyl p53 Arg peptide that was incubated with Sir2Tm and [³²P]NAD⁺. The sites of ribosylation were determined by relating the Edman degradation cycle to the known protein sequence. Error bars indicate average deviation.

FIGURE 4.

Structure of the Sir2Tm acetyl +2 Arg (+2R) p53 peptide. The structure of the Sir2Tm-acetyl p53 Arg peptide complex was determined to 1.5 Å resolution. A, the 2F_o − F_c electron density map (1 σ). B, the Sir2Tm (gray) Asp-49 contact with the acetyl p53 Arg (yellow). C, superposition of the Sir2Tm-S-alkylamidate intermediate (purple) and Sir2Tm (gray)-acetyl p53 Arg (yellow) structures.

FIGURE 5.

Model of a Sir2Tm acetyl p53 Arg-S-alkylamidate complex. The Sir2Tm S-alklyl amidate-p53 +2 Arg (+2R) peptide complex was modeled to determine whether the arginine could approach the intermediate.

FIGURE 6.

Structure-based mechanism of acetyl-dependent Sir2Tm-mediated ribosylation. In the first step of the reaction (step I), the acetyl lysine reacts with NAD⁺ to generate the O-alkylamidate. The O-alkylamidate can react with nicotinamide to regenerate the starting reactants, NAD⁺ and acetyl lysine, react with the 2′OH from the nicotinamide-ribose, leading to deacetylation products, or react with the arginine, resulting in ribosylation of the peptide substrate (step II).

FIGURE 7.

Model of Sir2Tm bound to deacetylated peptide and NAD⁺. A model based on the Sir2Tm deacetylated peptide of Sir2Tm bound to deacetylated p53 peptide and NAD⁺ was built using the Quanta software package (Accelerys). The peptide lysine and NAD⁺ are colored in yellow, and the conserved histidine 116 is colored in gray.