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. 2009 Dec;276(23):7159-76.
doi: 10.1111/j.1742-4658.2009.07427.x. Epub 2009 Nov 6.

Side chain specificity of ADP-ribosylation by a sirtuin

Kamau Fahie  1 , Po Hu, Stephen Swatkoski, Robert J Cotter, Yingkai Zhang, Cynthia Wolberger
Affiliations

Side chain specificity of ADP-ribosylation by a sirtuin

Kamau Fahie et al. FEBS J. 2009 Dec.

Abstract

Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical/molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated.

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Figures

Figure 1
Figure 1
TbSIR2 ADP-ribosylates unacetylated H1.1. (A) Autoradiograph of the enzyme-dependent ADP-ribosylation of unacetylated H1.1. Reactions were carried out using wild type TbSIR2. The catalytic mutants were used as control reactions. (B) 32P-incorporation into unacetylated H1.1 was determined for wild type TbSIR2 and the catalytic mutant TbH142Y. (C) TbSIR2 ADP-ribosylation of unacetylated H1.1 displays concentration dependent inhibition with nicotinamide. (D) Phosphodiesterase treatment removes the radiolabel from unacetylated H1.1 treated with TbSIR2 and 32P-NAD+. (E) Autoradiograph of ADP-ribosylation reactions using acetylated and unacetylated histone H1.1 as substrates.
Figure 2
Figure 2
TbSIR2 preferentially labels arginine containing peptides. (A) Sequence alignment of bovine histone H1.1 with chicken histone H1.11L. Chicken histone H1.11L is the closest homolog to bovine H1.1, whose structure has been partially determined. The identical residues are highlighted. The peptides used in ADP-ribosylation assays are underlined on the sequence of bovine H1.1. (B) Peptides which exhibited robust labeling from the microarray were used in solution-based assays. Peptide P19 was used as a negative control. (C) Amino acid substitutions of peptide P12 were used to determine the identity of the ADP-ribose acceptor. A double arginine variant of P12 was also tested. (D) ADP-ribosylation of the variant peptides was quantified using densitometry. (E) Autoradiograph of the ADP-ribosylation reactions of a poly-lysine (P12pK) and a poly-arginine (P12pR) peptide.
Figure 3
Figure 3
MALDI mass spectrometry confirms the incorporation of ADP-ribose in peptide substrates. (A) Peptides P12 and P39 were ADP-ribosylated in a non-radioactive assay. The treated peptides were analyzed by MALDI mass spectrometry. Samples treated with TbSIR2 show mass changes of 540 Daltons, consistent with the addition ADP-ribose moieties to the peptides. (B) MALDI mass spectrometry analysis of the ADP-ribosylation of peptide P12pR. The reactions were carried out using 20 μM peptide with 100 μM NAD+. Detection of the ADP-ribosylated P12pR was improved using higher concentrations of peptide and NAD+.
Figure 4
Figure 4
Arginine is the major ADP-ribose acceptor for unacetylated histone H1.1, but is not the major ADP-ribose acceptor for acetylated histone H1.1. (A) Nonenzymatic ADP-ribosylation of peptides P12pR and P12pK, generating an ADP-ribosylarginine substrate and an ADP-ribosyllysine substrate. (B) Enzymatic detection of ADP-ribosylarginine. Human ADP-ribosylarginine hydrolase (hADPRH) exhibits specificity for ADP-ribosylarginine, cleaving ADP-ribose from arginine side chains but not from lysine side chains. Phosphodiesterase I hydrolyzes the pyrophosphate bond for ADP-ribosylarginine and ADP-ribosyllysine. (C) Unacetylated and acetylated histone H1.1 were modified in ADP-ribosylation reactions then treated with hADPRH.
Figure 5
Figure 5
Reactant complex model for QM/MM calculations. (A) Substrate arginine binding in Sir2Tm enzyme. The overall substrate peptide binding pattern is very similar to that with acetyl-lysine substrate. (B) Illustration of the QM/MM partition in Sir2Tm ribosylation. Color notations: blue, QM subsystem; red, QM/MM boundary atoms described by improved pseudobond parameters; green, C1′-N1 bond; black, MM subsystem. (C) Proposed arginine ADP-ribosylation mechanism.
Figure 6
Figure 6
(A) Two-dimensional minimum energy surface along C1′–N1 and Nζ–C1′ bonds. The reactant is on the lower left corner, the product is on the upper right corner, and the transition state is well located in the middle. (B) Potential of mean force (PMF) for the Sir2Tm catalyzed ribosylation determined with B3LYP/6-31G(d) QM/MM MD simulations. The active site geometries at reactant, transition state, and product and some key bond lengths are also shown.
Figure 7
Figure 7
Computed group partial charges of arginine, nicotinamide, and ribose at reactant, transition state, and product. The di-phosphate portion has almost the constant partial charge of -2.10e during the reaction.
Figure 8
Figure 8
Proposed mechanisms for deacetylation and ADP-ribosylation catalyzed by TbSIR2. (A) Deacetylation-independent ADP-ribosylation involving the direct transfer of the ADP-ribose moiety to arginine. (B) Proposed mechanism of the NAD+-dependent deacetylation reaction. (C) Deacetylation-dependent ADP-ribosylation in which the O-alkylamidate intermediate reacts with a neighboring protein nucleophile.
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