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ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)–dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (MgA and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.

Graphical abstract, ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules controlling major biological processes as diverse as DNA damage repair, transcriptional regulation, intracellular transport, immune and stress responses, cell survival and proliferation. Furthermore, enzymatic reactions of ADPr are central in the pathogenesis of many human diseases, including infectious conditions. By providing a review of ADPr signalling in bacterial systems, we highlight the relevance of this chemical modification in the pathogenesis of human diseases depending on host-pathogen interactions. The post-antibiotic era has raised the need to find alternative approaches to antibiotic administration, as major pathogens becoming resistant to antibiotics. An in-depth understanding of ADPr reactions provides the rationale for designing novel antimicrobial strategies for treatment of infectious diseases. In addition, the understanding of mechanisms of ADPr by bacterial virulence factors offers important hints to improve our knowledge on cellular processes regulated by eukaryotic homologous enzymes, which are often involved in the pathogenesis of human diseases.