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

Abstract

Posttranslational 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 apoptosis1. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesised from NAD by poly(ADP-ribose) polymerases (PARPs)1,2. Poly(ADP-ribose) glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death3,4. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that exhibits 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 macro domain family5,6. 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. Our 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 link to human disease.

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

“…Thus far, we have not been able to experimentally demonstrate the endoglycohydrolase activity for any of our PARG enzyme preparations and we consistently observed ADP-ribose as the only detectable product ( Supplementary Fig. S8) 10 . Nonetheless, we tested some mutants at the n + 1 ADP-ribose binding site that, according to our PAR-PARG model, could specifically affect the endo-glycohydrolase function.…”

“…NAMD software 23 was used to perform all molecular dynamics (MD) simulations of the ADP-α-ribose tetramer in complex with PARG 10 . PAR tetramer was created by addition of three additional ADP-a-ribose monomers to the ADP-a-ribose bound to PARG.…”

“…The disruption of the PARG gene in mouse and Drosophila melanogaster leads to early embryonic lethality 8,9 . A highly diverged PARG (bacterial-type) is present in filamentous fungi and a number of bacterial species 10 . The recently solved structure of a bacterial PARG revealed that the catalytic centre is essentially a macrodomain with a loop region inserted that contains the PARG signature sequence (GGG-X 6 − 8 -QEE) 5,10 .…”

“…A highly diverged PARG (bacterial-type) is present in filamentous fungi and a number of bacterial species 10 . The recently solved structure of a bacterial PARG revealed that the catalytic centre is essentially a macrodomain with a loop region inserted that contains the PARG signature sequence (GGG-X 6 − 8 -QEE) 5,10 . Macrodomains are evolutionarily conserved ADP-ribose-binding modules that often mediate PAR signalling [11][12][13] .…”

“…Macrodomains are evolutionarily conserved ADP-ribose-binding modules that often mediate PAR signalling [11][12][13] . The bacterial PARG structures combined with biochemical studies established a role for the PARG signature residues in substrate binding and catalysis 10 . Despite this progress, structural information on the canonical PARGs is still lacking.…”