A family of macrodomain proteins reverses cellular mono-ADP-ribosylation

Abstract

ADP-ribosylation is a reversible post-translational modification with wide-ranging biological functions in all kingdoms of life. A variety of enzymes use NAD(+) to transfer either single or multiple ADP-ribose (ADPr) moieties onto distinct amino acid substrates, often in response to DNA damage or other stresses. Poly-ADPr-glycohydrolase readily reverses poly-ADP-ribosylation induced by the DNA-damage sensor PARP1 and other enzymes, but it does not remove the most proximal ADPr linked to the target amino acid. Searches for enzymes capable of fully reversing cellular mono-ADP-ribosylation back to the unmodified state have proved elusive, which leaves a gap in the understanding of this modification. Here, we identify a family of macrodomain enzymes present in viruses, yeast and animals that reverse cellular ADP-ribosylation by acting on mono-ADP-ribosylated substrates. Our discoveries establish the complete reversibility of PARP-catalyzed cellular ADP-ribosylation as a regulatory modification.

Figure 1. Macrodomain proteins interact with and demodify weakly ADP-ribosylated PARP1.

(a) Recruitment of tagged mEGFP-MacroD2 and mCherry-MacroH2A.1.1 macrodomains to sites of laser-induced DNA damage. The focus of laser microirradiation is indicated with a yellow circle. Scale bar, 10 μm. (b) Anti-PAR (10H) western blot of poly-ADP-ribosylated PARP1 immunoprecipitates (IP). MacroD2 and MacroH2A.1.1 immunoprecipitation of highly (1 mM NAD⁺) or weakly (30 μM NAD⁺) poly-ADP-ribosylated PARP1. (c) Immunoprecipitation and anti-PAR (10H) western blot of PARP1 automodification reactions co-incubated with MacroD2 for the indicated times. (d) Autoradiography and quantification of PARP1 demodification reactions. PARP1 automodified with molar excess (left) or substoichiometric amounts (right) of NAD⁺ spiked with [³²P]NAD⁺, subjected to demodification by MacroD2, MacroH2A.1.1 or PARG are shown. The residual radioactivity is normalized to buffer control. Error bars, s.e.m. (n = 3). Conc, concentration.

Figure 2. Macrodomain proteins reversibly remove ADPr from ADP-ribosylated PARP10.

(a) Immunoprecipitates of GST-PARP10 catalytic domain and anti-GST western blot. Samples are of unmodified (−) or ADP-ribosylated (+) GST-PARP10 pulled down with MacroD2 wild type (WT), the O-acetyl-ADP-ribose hydrolysis–deficient double mutant N92A D102A and the ADPr binding–deficient mutant G188E. (b) Autoradiography and quantification of GST-PARP10 catalytic-domain demodification reactions. Samples are demodification reactions containing the indicated macrodomain proteins. The removed radioactive signal is normalized to the buffer control. Error bars, s.e.m. (n = 3). (c) UV shadowing and autoradiography of thin-layer chromatograph. Samples from b alongside with AMP, ADP, NAD⁺ and ADPr standards separated by thin-layer chromatography and visualized by UV shadowing or ³²P autoradiography are shown. (d) MALDI-MS analysis of PARP10 demodification reactions. Analysis of ADPr standard (top left), modified PARP10 (top right), modified PARP10 together with MacroD2 (bottom left) or nonmodified PARP10 together with MacroD2 (bottom right) samples. The 558.1 m/z peak corresponds to ADPr.

Figure 3. MacroD2 hydrolyzes mono-ADP-ribosylated PARP10 at the C1′′ atom of ADPr.

(a) Close-up view of the 1.5-Å-resolution X-ray structure of the MacroD2 macrodomain in complex with ADPr (gray), focusing on the distal ribose unit of ADPr. Residues in the vicinity of the distal ribose are shown in stick representation (purple). Hydrogen bonds between protein and ligand are indicated by dashed lines (blue). The positioned H₂O, coordinated by hydrogen bonds to the 1′′-OH, 5′′-O and α-phosphate, is shown (purple sphere). The regions forming the two sides of the distal ribose binding cleft are labeled loop 1 and 2. (b) ESI-MS analysis of PARP10 demodification reaction in the presence of H₂¹⁸O. Samples are PARP10 incubated with MacroD2 (top) or ADPr under the same reaction conditions (bottom) in the presence of 66% H₂¹⁸O. The 560.1 m/z peak indicates the ¹⁸O atom containing ADPr. (c) Snapshots from a molecular dynamics simulation of MacroD2 in complex with an ADPr-glutamate ester. The MacroD2 binding region is indicated as cartoon (pink), and the bound ADPr ester is shown as a stick model with the glutamate residue (arrow) adopting various conformations and orientations (1–3). A water molecule adopts the placement close to the presumed catalytically active water and is represented as van der Waals surface.

Figure 4. The mode of ADPr coordination in the macrodomain pocket determines mono-ADP-ribosyl hydrolase function and identifies evolutionarily conserved enzymes.

(a–c) Superposition of MacroD2 with A. fulgidus AF1521 (PDB 2BFQ) (a), T. curvata PARG (PDB 3SIG), with the catalytic residue Glu115 unique to PARG labeled (b) or MacroH2A.1.1 (PDB 3IID) (c). Isostructural residues and conserved water molecules are highlighted in red. The positions of the 1′′-OH groups of ADPr are indicated. (d) Quantification of macrodomain proteins' demodification activity on [³²P]ADP-ribosylated PARP10. Samples include either MacroD2, MacroH2A.1.1, AF1521, PARG, Poa1p or Ymx7 macrodomains. Prefix indicates organism: Af, A. fulgidus; Hs, human, Sc, Saccharomyces cerevisiae. ³²P removal is normalized to buffer control. (e) Structure-guided sequence alignment of selected macrodomains. Two sequence regions around loop 1 and 2 are shown. Secondary structure elements are taken from the MacroD2 structure. Signature motif residues important for the coordination of the ADPr distal ribose and indicative for hydrolysis activity are highlighted in red. Plus indicates confirmed hydrolysis activity; minus indicates no activity; asterisks highlight the residues most important for catalytic activity.