The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

Abstract

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Figure 1. Phylogeny and functional relationship between DUF2263 and canonical-type PARGs

a, Multiple sequence alignment of different DUF2263 and PARG proteins from Thermomonospora curvata (Tcu), Herpetosiphon aurantiacus (Hau), Deinococcus radiodurans (Dra), Aspergillus fumigatus (Afu), Homo sapiens (Hsa), Bos taurus (Bta) and Entamoeba dispar (Edi). The two catalytic glutamates, a conserved glycine and tyrosine are marked with black asterisks, grey asterisk and black cross respectively. Secondary structure elements from the Tcu PARG structure are indicated above. b, YmdB-rooted phylogenetic tree of PARGs implied by the neighbour-joining method. Organisms devoid of PARP are marked in grey. c, H. aurantiacus (HA) PARP and PARG enzymes are active as shown by Western blotting with anti-PAR antibodies.

Figure 2. Poly(ADP-ribose) hydrolytic activities of divergent and canonical PARGs

a, A colorimetric PARG assay. b, Analysis of the hydrolysis of the PARP1-generated PAR substrate by anti-PAR antibodies ‘AF’ stands for A. fumigatus; ‘DR’ D. radiodurans; ‘HA’ H. aurantiacus; ‘TC’ T. curvata; ‘AV’ A. variabilis; ‘HS’ H. sapiens; ‘ED’ E. dispar. c, SDS-PAGE-based assay with [³²P]-automodified PARP1 substrate. PARGs are inhibited by ADP-HPD. d, TLC analysis of PARG activity on the [³²P]-PAR substrate. The right side of the TLC plate was visualised by shadowing under UV. e, Heterologously expressed A. fumigatus and D. radiodurans PARG hydrolyse PAR in hPARP1-expressing budding yeast cells.

Figure 3. T. curvata PARG crystal structure in complex with ADP-ribose

a, b, Overall fold of PARG with α-helices depicted in red, β-sheets in blue and bound ADP-ribose in green spheres. The PARG-specific catalytic loop is shown in yellow, and the diphosphate-binding loop in magenta. c, Overlay of the T. curvata PARG structure with a representative macro domain structure (PDB code 2BFQ, in cyan). The PARG-specific N-terminal extension and additional PARG structural elements are highlighted in red. Structural features that are similar to 2BFQ and other macro domains are shown in gray. d, Detailed view of ADP-ribose bound in the PARG active site. ADP-ribose is shown with the corresponding 2F_o-F_c omit-map density contoured at 1.2 σ in blue. Key active site residues are represented in atom coloured sticks with hydrogen bonds indicated by black dotted lines.

Figure 4. Structural basis of poly(ADP-ribose) glycohydrolysis

a, The PARG solvent accessible surface derived from molecular dynamic simulations b, A detailed view of the lowest energy PARG:ADP-ribose-dimer model obtained from the 100 snapshot structures. Key active site residues are highlighted in green with PAR structural elements coloured as in 4a. c, Proposed mechanism for PAR glycohydrolysis. R₁ and R₂ represent (n-1) PAR and terminal adenosine moieties respectively. d, PARG activities of the T. curvata PARG mutants and the corresponding H. sapiens PARG mutants. Error bars represent s.d. (n=3).